Preparation of Templates for Nucleic Acid Sequencing

ABSTRACT

The invention relates to methods of generating templates for a nucleic acid sequencing reaction which comprise:
         providing at least one double-stranded nucleic acid molecule, wherein both strands of the double-stranded nucleic acid molecule are attached to a solid support at the 5′ end,   cleaving one or both strands of the double-stranded nucleic acid molecule, and   subjecting the cleaved strand(s) to denaturing conditions to remove the portion of the cleaved strand(s) not attached to the solid support, thereby generating a partially or substantially single-stranded template for a nucleic acid sequencing reaction,

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 11/989,169,filed Dec. 22, 2008, which is a National Stage Application of PCTApplication No. PCT/GB2006/002687, filed Jul. 20, 2006, which in turn,claims priority from Great Britain Application Serial No. GB 0514936.4,filed Jul. 20, 2005. The entire disclosures of these applications areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to the preparation of templates for nucleic acidsequencing reactions and to methods of sequencing such templates. Inparticular, the invention relates to the preparation of template nucleicacid molecules ready for sequencing by cleavage of one or both strandsof a double-stranded nucleic acid immobilised on a solid support.

BACKGROUND TO THE INVENTION

Nucleic acid sequencing methods have been known in the art for manyyears. One of the best-known methods is the Sanger “dideoxy” methodwhich relies upon the use of dideoxyribonucleoside triphosphates aschain terminators. The Sanger method has been adapted for use inautomated sequencing with the use of chain terminators incorporatingfluorescent labels.

There are also known in the art methods of nucleic acid sequencing whichare based on successive cycles of incorporation of fluorescentlylabelled nucleic acid analogues. In such “sequencing by synthesis” or“cycle sequencing” methods the identity of the added base is determinedafter each nucleotide addition by detecting the fluorescent label.

In particular, U.S. Pat. No. 5,302,509 describes a method for sequencinga polynucleotide template which involves performing multiple extensionreactions using a DNA polymerase or DNA ligase to successivelyincorporate labelled polynucleotides complementary to a template strand.In such a “sequencing by synthesis” reaction a new polynucleotide strandbased-paired to the template strand is built up in the 5′ to 3′direction by successive incorporation of individual nucleotidescomplementary to the template strand. The substrate nucleosidetriphosphates used in the sequencing reaction are labelled at the 3′position with different 3′ labels, permitting determination of theidentity of the incorporated nucleotide as successive nucleotides areadded.

In order to maximise the throughput of nucleic acid sequencing reactionsit is advantageous to be able to sequence multiple template molecules inparallel. Parallel processing of multiple templates can be achieved withthe use of nucleic acid array technology. These arrays typically consistof a high-density matrix of polynucleotides immobilised onto a solidsupport material.

Various methods for fabrication of arrays of immobilised nucleic assayshave been described in the art. Of particular interest, WO 98/44151 andWO 00/18957 both describe methods of nucleic acid amplification whichallow amplification products to be immobilised on a solid support inorder to form arrays comprised of clusters or “colonies” formed from aplurality of identical immobilised polynucleotide strands and aplurality of identical immobilised complementary strands. Arrays of thistype are referred to herein as “clustered arrays”. The nucleic acidmolecules present in DNA colonies on the clustered arrays preparedaccording to these methods can provide templates for sequencingreactions, for example as described in WO 98/44152.

The products of solid-phase amplification reactions such as thosedescribed in WO 98/44151 and WO 00/18957 are so-called “bridged”structures formed by annealing of pairs of immobilised polynucleotidestrands and immobilised complementary strands, both strands beingattached to the solid support at the 5′ end. Arrays comprised of suchbridged structures provide inefficient templates for nucleic acidsequencing, since hybridisation of a conventional sequencing primer toone of the immobilised strands is not favoured compared to annealing ofthis strand to its immobilised complementary strand under standardhybridisation conditions.

In order to provide more suitable templates for nucleic acid sequencingit is preferred to remove substantially all or at least a portion of oneof the immobilised strands in the “bridged” structure in order togenerate a template which is at least partially single-stranded. Theportion of the template which is single-stranded will thus be availablefor hybridisation to a sequencing primer. The process of removing all ora portion of one immobilised strand in a “bridged” double-strandednucleic acid structure may be referred to herein as “linearisation”.

It is known in the art that bridged template structures may belinearised by cleavage of one or both strands with a restrictionendonuclease. A disadvantage of the use of restriction enzymes forlinearisation is that it requires the presence of a specific recognitionsequence for the enzyme at a suitable location in the bridged templatestructure. There is a risk that the same recognition sequence may appearelsewhere in the bridged structure, meaning that the enzyme may cut atone or more further sites, in addition to the intended cleavage site forlinearisation. This may be a particular problem where the bridgedstructures to be linearised are derived by solid-phase amplification oftemplates of partially unknown sequence, since it cannot be predicted inadvance whether a particular enzyme will cut within the region ofunknown sequence.

Therefore, in one general aspect the invention provides methods oftemplate linearisation which do not require cleavage with restrictionendonucleases, or with nicking endonucleases.

In another general aspect the invention relates to methods of templatelinearisation which are compatible with a particular type of solidsupported microarray. More specifically, the invention provideslinearisation methods which are compatible with arrays formed on solidsupported polyacrylamide hydrogels.

In preparing hydrogel-based solid-supported molecular arrays, a hydrogelis formed and molecules displayed from it. These two features—formationof the hydrogel and construction of the array—may be effectedsequentially or simultaneously. Where the hydrogel is formed prior toformation of the array, it is typically produced by allowing a mixtureof co-monomers to polymerise. Generally, the mixture of co-monomerscontain acrylamide and one or more co-monomers, the latter of whichpermit, in part, subsequent immobilisation of molecules of interest soas to form the molecular array.

The co-monomers used to create the hydrogel typically contain afunctionality that serves to participate in crosslinking of the hydrogeland/or immobilise the hydrogel to the solid support and facilitateassociation with the target molecules of interest.

The present inventors have shown that clustered arrays may be formed onsuch solid-supported hydrogels by solid phase nucleic acid amplificationusing forward and reverse amplification primers attached to the hydrogelat their 5′ ends, leading to the production of clustered arrays ofamplification products having a “bridged” structure. In order tomaximise the efficiency of sequencing reactions using templates derivedfrom such bridged products there is a need for linearisation methodswhich are compatible with the hydrogel surface and with subsequentnucleic acid sequencing reactions.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method of generating atemplate for a nucleic acid sequencing reaction comprising,

(i) providing a solid supported polyacrylamide hydrogel having attachedthereto one or more double-stranded nucleic acid molecules, wherein bothstrands of the double-stranded nucleic acid molecule are attached to thepolyacrylamide hydrogel at the 5′ end,(ii) cleaving one or both strands of the double-stranded nucleic acidmolecule, and(iii) subjecting the cleaved strand(s) to denaturing conditions toremove the portion of the cleaved strand(s) not attached to thepolyacrylamide hydrogel, thereby generating a partially or substantiallysingle-stranded template for a nucleic acid sequencing reaction.

In a preferred embodiment of this aspect of the invention step (ii) doesnot comprise cleavage with a restriction endonuclease or a nickingendonuclease.

In a second aspect the invention provides method of generating atemplate for a nucleic acid sequencing reaction comprising,

(i) providing at least one double-stranded nucleic acid molecule,wherein both strands of the double-stranded nucleic acid molecule areattached to a solid support at the 5′ end,(ii) cleaving one or both strands of the double-stranded nucleic acidmolecule, and(iii) subjecting the cleaved strand(s) to denaturing conditions toremove the portion of the cleaved strand(s) not attached to the solidsupport, thereby generating a partially or substantially single-strandedtemplate for a nucleic acid sequencing reaction,characterised in that step (ii) does not comprise cleavage with arestriction endonuclease or a nicking endonuclease.

In one embodiment of both the first and second aspects of the inventionthe double-stranded stranded nucleic acid molecule may be cleaved at apre-determined cleavage site. By “pre-determined” cleavage site is meanta site whose location is determined in advance of the cleavage reaction,as opposed to cleavage at a random site the location of which is notknown in advance.

In one embodiment of both the first and second aspects of the inventioncleavage may occur at a cleavage site in one or both strands of thedouble-stranded nucleic acid molecule which comprises one or more or anycombination of non-natural nucleotides, ribonucleotides or anon-nucleotide chemical modifications. The position of this cleavagesite is preferably pre-determined.

In one embodiment of both the first and second aspects of the inventionthe double-stranded nucleic acid molecule may be cleaved in one or bothstrands via a non-enzymatic chemical cleavage reaction. In a specificnon-limiting embodiment one strand of the double-stranded nucleic acidmolecule may comprise a diol linker and this strand may be cleaved bytreatment with periodate.

In a further embodiment of both the first and second aspects of theinvention one strand of the double-stranded nucleic acid molecule may betreated to generate an abasic site and then cleaved at the abasic site.In a non-limiting specific embodiment, wherein one strand of thedouble-stranded nucleic acid molecule includes a uracil base, the abasicsite may be generated by treatment with uracil DNA glycosylase and thencleaved with endonuclease, heat treatment or alkali treatment.

In a further embodiment of both the first and second aspects of theinvention one strand of the double-stranded nucleic acid may compriseone or more ribonucleotides and step (ii) may comprise cleaving thisstrand adjacent to a ribonucleotide using an RNAse or a non-enzymaticchemical cleavage agent. Suitable non-enzymatic chemical cleavage agentsinclude metal ions, and in particular rare earth metal ions, e.g. La³⁺or Lu³⁺.

In a further embodiment of both the first and second aspects of theinvention one strand of the double-stranded nucleic acid may compriseone or more methylated nucleotides and step (ii) may comprise cleavingthis strand using an enzyme specific for a recognition sequenceincluding said methylated nucleotide(s).

In a further embodiment of both the first and second aspects of theinvention step (ii) may comprise cleaving one or both strands of thedouble-stranded nucleic acid in a photochemical reaction.

In a further embodiment of the first and second aspects of the inventionone strand of the double-stranded nucleic acid molecule may have apeptide covalently linked at the 5′ end and step (ii) may comprisecleaving the peptide.

In a third aspect the invention also provides methods of sequencingnucleic acid templates generated according to the methods of the firstand second aspects of the invention.

The present invention will now be further described. In the followingpassages different features of the various aspects of the invention aredefined in more detail. Each feature so defined in connection with oneaspect of the invention may be combined with features described inconnection with any other aspect of the invention unless clearlyindicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature orfeatures indicated as being preferred or advantageous.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a schematic illustration of cluster formation by solid-phasePCR and subsequent linearisation and annealing of a sequencing primer.The starting material in step (a) is a solid support grafted with amixture of amplification primers, one of which comprises a cleavagesite. The primers are covalently attached to the solid support at the 5′end. A substrate molecule to be amplified is also applied to the solidsupport, either by hybridisation to one of the immobilised primers or bydirect covalent attachment to the support at the 5′ end. FIG. 1( b)schematically illustrates the “bridged” amplification products resultingfrom solid phase amplification. For simplicity only a small number ofbridged products are shown. The amplification products are then“linearised” by cleavage at the cleavage sites derived from theamplification primers (FIG. 1( c)). The products of the cleavagereaction may then be subjected to denaturing conditions, resulting inremoval of the portions of the cleaved strands which are no longercovalently attached to the solid support (FIG. 1( d)). The remainingsingle stranded products may then be hybridised to a sequencing primer(FIG. 1( e)).

FIG. 2. is a schematic illustration of linearisation by cleavage at anabasic site generated by treatment of a U-containing polynucleotide withuracil DNA glycosylase. Bridged amplification products are generated byamplification of the template structure schematically illustrated inFIG. 2( a) with primers immobilised on a solid support. One of theamplification primers contains a deoxyuridine nucleotide represented asU. A single bridged product is illustrated in FIG. 2( b) for simplicity.An abasic site is generated in one strand by treatment with uracil DNAglycosylase. This strand may then be cleaved by hydrolysis of the abasicsite to generate the “nicked” structure illustrated in FIG. 2( c).Hydrolysis of the abasic site generates a free 3′ hydroxyl group whichcan serve as an initiation point for a sequencing reaction. Sequencingmay be carried out using a strand displacing polymerase (as shown inFIG. 2( d), or the portion of the cleaved strand not attached to thesolid support may be removed by denaturation prior to sequencing. In thelatter case the sequencing reaction could be initiated by hybridisationof a sequencing primer as an alternative to priming from the cleavedstrand itself.

FIG. 3. illustrates linearisation by cleavage with a restriction enzyme.FIG. 3( a) shows the sequence of a representative double-strandednucleic acid molecule (top strand SEQ ID NO:9; bottom strand SEQ IDNO:10) which includes sequences derived from the amplification primersP5 and P7 utilised in the accompanying examples, a restriction site forcleavage by the enzyme BglII and a site for binding of a sequencingprimer. “Genomic” represents a sequence derived from a genomic DNAfragment. The genomic fragment will typically be from 400-700b inlength, although the invention is not limited to sequences of thislength, and may be of known, partially known or unknown sequence. FIG.3( b) schematically illustrates linearisation of a single bridgeddouble-stranded nucleic acid product having the sequence shown in FIG.3( a). The two complementary strands forming the bridged product arecovalently attached to a solid support at their 5′ ends. The bridgedproduct is first cleaved with BglII and then denatured to removesubstantially all of one strand. The single strand remaining on thesolid support may then be hybridised to a sequencing primer.

FIG. 4. graphically illustrates cleavage of a diol linker by treatmentwith sodium periodate.

FIG. 5. shows signal intensity versus time for SyBr green stainedcolonies treated with periodate, illustrating selective cleavage ofcolonies containing diol linkers.

FIG. 6. graphically illustrates cleavage of colonies containing diollinkers.

FIG. 7. is a schematic representation of an 8 channel flowcell suitablefor carrying out the methods of the invention.

FIG. 8. illustrates the structure and sequence of an exemplary DNAtemplate (top strand, SEQ ID NO:8; bottom strand, SEQ ID NO:11) used forsolid-phase PCR amplification in the accompanying examples. Sequences ofthe amplification primers P5 and P7 are shown in bold type. Thesequencing primer for the insert is also shown (SEQ ID NO:7).

FIG. 9. shows fluorescent CCD images of clustered arrays of nucleic acidcolonies following a single cycle of nucleotide incorporation understandard sequencing conditions. Panel (A) illustrates nucleotideincorporation on colonies of nucleic acids which have not beenlinearised. Pnale (B) illustrates nucleotide incorporation of coloniesof nucleic acid templates which have been linearised by periodatecleavage of a diol linker.

FIG. 10. shows histogram plots (fluorescence intensity vs number ofcolonies) corresponding to the fluorescent CCD images shown in FIG. 9.

DETAILED DESCRIPTION OF THE INVENTION

In its various aspects the invention generally relates to methods offorming templates for nucleic acid sequencing starting fromdouble-stranded nucleic acid molecules immobilised on a solid support,and to methods of sequencing such templates.

The double-stranded nucleic acid molecules which provide the startingpoint for sequencing template formation according to the first andsecond aspects of the invention are characterised in that they areformed from annealed complementary nucleic acid strands that areattached to the solid support at their 5′ ends, preferably via covalentattachment. When the complementary strands of the double-strandednucleic acid molecule are annealed, such as will generally be the casewhen the molecules are maintained under non-denaturing conditions, suchmolecules may be referred to herein as “bridged” structures.

The methods of template formation provided by the invention involvecleavage of one or both strands of the double-stranded molecule.Following the cleavage step the cleaved products may be subjected todenaturing conditions so as to remove the portion(s) of the cleavedstrand(s) which are not attached to the solid support, i.e. the portionlocated downstream of the site of cleavage when a given strand is viewed5′ to 3′.

The resulting template molecule will be at least partiallysingle-stranded and may be substantially single-stranded. The length ofthe single-stranded portion will depend on the position of the cleavagesite relative to the 5′ ends of the complementary strands and whetherthe cleavage step cuts one or both strands. It will be appreciated thatthe location of the cleavage site determines how much of each strandremains attached to the solid support after cleavage and denaturation.

The double-stranded nucleic acid molecule from which the sequencingtemplate is to be derived comprises two annealed (complementary)polynucleotide strands which are both attached to a solid support at ornear the 5′ end. Linkage to the solid support will preferably be viacovalent attachment. It will be appreciated that the annealed strandsneed not necessarily be fully complementary along their entire length.

When referring to attachment of molecules (e.g. nucleic acids) to asolid support, the terms “immobilised” and “attached” are usedinterchangeably herein and both terms are intended to encompass director indirect, covalent or non-covalent attachment, unless indicatedotherwise, either explicitly or by context. In certain embodiments ofthe invention covalent attachment may be preferred, but generally allthat is required is that the molecules (e.g. nucleic acids) remainimmobilised or attached to the support under the conditions in which itis intended to use the support, for example in applications requiringnucleic acid amplification and/or sequencing.

Certain embodiments of the invention make use of solid supportscomprised of an inert substrate or matrix (e.g. glass slides, polymerbeads etc) which is been “functionalised”, for example by application ofa layer or coating of an intermediate material comprising reactivegroups which permit covalent attachment to biomolecules, such aspolynucleotides. Examples of such supports include, but are not limitedto, polyacrylamide hydrogels supported on an inert substrate such asglass. In such embodiments, the biomolecules (e.g. polynucleotides) maybe directly covalently attached to the intermediate material (e.g. thehydrogel) but the intermediate material may itself be non-covalentlyattached to the substrate or matrix (e.g. the glass substrate). The term“covalent attachment to a solid support” is to be interpretedaccordingly as encompassing this type of arrangement.

In all aspects of the invention, covalent attachment can be achievedthrough a sulphur-containing nucleophile, such as phosphorothioate,present at the 5′ end of a polynucleotide strand. In the case of arraysbased on solid-supported polyacrylamide hydrogels, this nucleophile willbind to a “C” group present in the hydrogel.

The “double-stranded” nucleic acid to be cleaved may in fact bepartially single-stranded at the 5′ end(s) of one or both strands. Aswill be discussed in further detail hereinbelow, the double-strandednucleic acid will typically be formed from two complementarypolynucleotide strands comprised of deoxyribonucleotides joined byphosphodiester bonds, but may additionally include one or moreribonucleotides and/or non-nucleotide chemical moieties and/ornon-naturally occurring nucleotides and/or non-naturally occurringbackbone linkages. In particular, the double-stranded nucleic acid mayinclude non-nucleotide chemical moieties, e.g. linkers or spacers, atthe 5′ end of one or both strands. By way of non-limiting example, thedouble-stranded nucleic acid may include methylated nucleotides, uracilbases, phosphorothioate groups, also peptide conjugates etc. Suchnon-DNA or non-natural modifications may be included in order to permitcleavage, or to confer some other desirable property, for example toenable covalent attachment to a solid support, or to act as spacers toposition the site of cleavage an optimal distance from the solidsupport.

The site for cleavage of a strand of the double-stranded nucleic acidmay, depending on the nature of the cleavage reaction, be positioned ina region of the molecule that is single-stranded when the complementarystrands are annealed. As outlined above, the double-stranded nucleicacid may in fact be partially single-stranded at one or both 5′ ends,e.g. proximal to the site of linkage to the solid support. It is withinthe scope of the invention for a cleavage site to be positioned withinsuch a single-stranded region. In other embodiments the cleavage sitemay be present in a non-nucleotide chemical moiety covalently attachedto the 5′ end of one strand of the double-stranded nucleic acid, forexample a linker moiety.

The double-stranded nucleic acid will typically comprise a “target”region that it is desired to fully or partially sequence. The nature ofthe target region is not limiting to the invention. It may be ofpreviously known or unknown sequence and may be derived, for example,from a genomic DNA fragment, a cDNA, etc. The double-stranded nucleicacid molecule may also include non-target sequences, for example at the5′ and 3′ ends of both strands, flanking the target region. If thedouble-stranded nucleic acid is formed by solid-phase nucleic acidamplification, these non-target sequences may be derived from theprimers used for the amplification reaction. Sites for cleavage of oneor both strands of the double-stranded nucleic acid may be positioned inthe non-target sequences.

The double-stranded nucleic acid may form part of a cluster or colonycomprised of many such double-stranded nucleic acid molecules, and thecluster or colony may itself form part of an array of such clusters orcolonies, referred to herein as a “clustered array”. On such an arrayeach double-stranded nucleic acid molecule within each colony willcomprise the same target region, whereas different colonies may beformed of double-stranded nucleic acid molecules comprising differenttarget regions. In a preferred embodiment at least 90%, more preferablyat least 95% of the colonies on a given clustered array will be formedfrom double-stranded nucleic acid molecules comprising different targetregions, although within each individual colony on the array alldouble-stranded nucleic acid molecules will comprise the same targetregion.

On such a clustered array it is preferred that all double-strandednucleic acid molecules within all of the colonies on the array comprisethe same type of cleavage site. This is preferred even when differentcolonies on the array are formed of double-stranded nucleic acidmolecules comprising different target regions, since it enables all thedouble-stranded molecules on the array to be cleaved simultaneouslyunder identical cleavage reaction conditions.

Cleavage Methods

Various cleavage methods may be used in accordance in accordance withthe first and second aspects of the invention to cleave one or bothstrands of the double-stranded nucleic acid molecule. Preferred butnon-limited embodiments of suitable cleavage methods are discussed infurther detail below. Methods i) to vii) are common to the first andsecond aspects of the invention.

i) Chemical Cleavage

The term “chemical cleavage” encompasses any method which utilises anon-nucleic acid and non-enzymatic chemical reagent in order topromote/achieve cleavage of one or both strands of the double-strandednucleic acid molecule. If required, one or both strands of thedouble-stranded nucleic acid molecule may include one or morenon-nucleotide chemical moieties and/or non-natural nucleotides and/ornon-natural backbone linkages in order to permit a chemical cleavagereaction at a pre-determined cleavage site.

In a preferred but non-limiting embodiment one strand of thedouble-stranded nucleic acid molecule may include a diol linkage whichpermits cleavage by treatment with periodate (e.g. sodium periodate).The diol linkage may be positioned at a pre-determined cleavage site,the precise location of which may be selected by the user. It will beappreciated that more than one diol could be included at the cleavagesite.

Diol linker units based on phosphoamidite chemistry suitable forincorporation into polynucleotide chains are commercially available fromFidelity systems Inc. (Gaithersburg, Md., USA). One or more diol unitsmay be incorporated into a polynucleotide using standard methods forautomated chemical DNA synthesis.

In order to position the diol linker at an optimum distance from thesolid support one or more spacer molecules may be included between thediol linker and the site of attachment to the solid support. The spacermolecule may be a non-nucleotide chemical moiety. Suitable spacer unitsbased on phosphoamidite chemistry for use in conjunction with diollinkers are also supplied by Fidelity Systems Inc. One suitable spacerfor use with diol linkers is the spacer denoted arm 26, identified inthe accompanying examples. arm 26 may be modified to include aphosphorothioate group at the 5′ end in order to facilitate attachmentof the 5′ end of the polynucleotide strand to a solid support. Thephosphorothioate group can easily be attached during chemical synthesisof a “polynucleotide” chain including the spacer and diol units.

Other spacer molecules could be used as an alternative to arm 26. Forexample, a stretch of non-target “spacer” nucleotides may be included.Typically from 1 to 20, more preferably from 1 to 15 or from 1 to 10,and more particularly 2, 3, 4, 5, 6, 7, 8, 9 or 10 spacer nucleotidesmay be included. Most preferably 10 spacer nucleotides will bepositioned between the point of attachment of the polynucleotide strandto a solid support (typically the extreme 5′ end) and the diol linker.It is preferred to use polyT spacers, although other nucleotides andcombinations thereof can be used. In one preferred embodiment the strandto be cleaved may include 10T spacer nucleotides upstream of the diollinker.

The diol linker is cleaved by treatment with a “cleaving agent”, whichcan be any substance which promotes cleavage of the diol. The preferredcleaving agent is periodate, preferably aqueous sodium periodate(NaIO₄). Following treatment with the cleaving agent (e.g. periodate) tocleave the diol, the cleaved product may be treated with a “cappingagent” in order to neutralise reactive species generated in the cleavagereaction. Suitable capping agents for this purpose include amines, suchas ethanolamine. Advantageously, the capping agent (e.g. ethanolamine)may be included in a mixture with the cleaving agent (e.g. periodate) sothat reactive species are capped as soon as they are formed.

The combination of a diol linkage and cleaving agent (e.g. periodate) toachieve cleavage of one strand of a double-stranded nucleic acidmolecule is preferred for linearisation of nucleic acid molecules onsolid supported polyacrylamide hydrogels because treatment withperiodate is compatible with nucleic acid integrity and with thechemistry of the hydrogel surface. However, the invention is notintended to be limited to the use of diol linkages/periodate as a methodof linearisation on polyacrylamide hydrogel surfaces but also extends touse of this cleavage method for linearisation of nucleic acidsimmobilised on other surfaces, including supports coated withfunctionalised silanes (etc).

ii) Cleavage Of Abasic Sites in a Double-Stranded Molecule

An “abasic site” is defined as a nucleoside position in a polynucleotidechain from which the base component has been removed. Abasic sites canoccur naturally in DNA under physiological conditions by hydrolysis ofnucleoside residues, but may also be formed chemically under artificialconditions or by the action of enzymes. Once formed, abasic sites may becleaved (e.g. by treatment with an endonuclease or other single-strandedcleaving enzyme, exposure to heat or alkali), providing a means forsite-specific cleavage of a polynucleotide strand.

In a preferred but non-limiting embodiment an abasic site may be createdat a pre-determined position on one strand of a double-strandedpolynucleotide and then cleaved by first incorporating deoxyuridine (U)at a pre-determined cleavage site in the double-stranded nucleic acidmolecule.

This can be achieved, for example, by including U in one of the primersused for preparation of the double-stranded nucleic acid molecule bysolid-phase PCR amplification. The enzyme uracil DNA glycosylase (UDG)may then be used to remove the uracil base, generating an abasic site onone strand. The polynucleotide strand including the abasic site may thenbe cleaved at the abasic site by treatment with endonuclease (e.g EndoIVendonuclease, AP lyase, FPG glycosylase/AP lyase, EndoVIIIglycosylase/AP lyase), heat or alkali.

Abasic sites may also be generated at non-natural/modifieddeoxyribonucleotides other than deoxyuridine and cleaved in an analogousmanner by treatment with endonuclease, heat or alkali. For example,8-oxo-guanine can be converted to an abasic site by exposure to FPGglycosylase. Deoxyinosine can be converted to an abasic site by exposureto AlkA glycosylase. The abasic sites thus generated may then becleaved, typically by treatment with a suitable endonuclease (e.g.EndoIV, AP lyase). If the double-stranded nucleic acid molecule isformed by solid-phase PCR amplification using an amplification primerwhich comprises the relevant non-natural/modified nucleotide, then it isessential in this embodiment that the non-natural/modified nucleotide iscapable of being copied by the polymerase used for the amplificationreaction.

In one embodiment, the double-stranded nucleic acid molecules to becleaved may be exposed to a mixture containing the appropriateglycosylase (to generate the abasic site) and one or more suitableendonucleases (to subsequently cleave). In such mixtures the glycosylaseand the endonuclease will typically be present in an activity ratio ofat least about 2:1.

Cleavage of double stranded nucleic acids at pre-determined abasic siteshas particular advantages in relation to the creation of templates fornucleic acid sequencing. In particular, cleavage at an abasic sitegenerated by treatment with a glycosylase such as UDG generates a free3′ hydroxyl group on the cleaved strand which can provide an initiationpoint for sequencing a region of the complementary strand. Moreover, ifthe starting double-stranded nucleic acid contains only one cleavablebase (e.g. uracil) on one strand then a single “nick” can be generatedat a unique position in this strand of the duplex. Since the cleavagereaction requires a residue, e.g. deoxyuridine, which does not occurnaturally in DNA, but is otherwise independent of sequence context, ifonly one non-natural base is included there is no possibility ofglycosylase-mediated cleavage occurring elsewhere at unwanted positionsin the duplex. In contrast, were the double-stranded nucleic acid to becleaved with a “nicking” endonuclease that recognises a specificsequence, there is a possibility that the enzyme may create nicks atother sites in the duplex (in addition to the desired cleavage site) ifthese possess the correct recognition sequence. This could present aproblem if nicks are created in the strand it is intended to sequencerather than the strand that will be fully or partially removed to createthe sequencing template and is a particular risk if the target portionof the double-stranded nucleic acid molecule is of unknown sequence.

The fact that there is no requirement for the non-natural residue (e.g.uracil) to be located in a detailed sequence context in order to providea site for cleavage using this approach is itself advantageous. Inparticular, if the cleavage site is to be incorporated into anamplification primer to be used in the production of a clustered arrayby solid-phase amplification, it is necessary only to replace onenatural nucleotide in the primer (e.g. T) with a non-natural nucleotide(e.g. U) in order to enable cleavage at a pre-determined cleavage site.There is no need to engineer the primer to include a restriction enzymerecognition sequence of several nucleotides in length. Oligonucleotideprimers including U nucleotides, and the other non-natural nucleotideslisted above, can easily be prepared using conventional techniques andapparatus for chemical synthesis of oligonucleotides.

Another advantage gained by cleavage of abasic sites in adouble-stranded molecule generated by action of UDG on uracil is thatthe first base incorporated in a “sequencing-by-synthesis” reactioninitiating at the free 3′ hydroxyl group formed by cleavage at such asite will always be T. Hence, if the double-stranded nucleic acidmolecule forms part of a clustered array comprised of many suchmolecules, all of which are cleaved in this manner to produce sequencingtemplates, then the first base universally incorporated across the wholearray will be T. This can provide a sequence-independent assay forcluster intensity at the start of a sequencing “run”.

iii) Cleavage of Ribonucleotides

Incorporation of one or more ribonucleotides into a polynucleotidestrand which is otherwise comprised of deoxyribonucleotides (with orwithout additional non-nucleotide chemical moieties, non-natural basesor non-natural backbone linkages) can provide a pre-determined site forcleavage using a chemical agent capable of selectively cleaving thephosphodiester bond between a deoxyribonucleotide and a ribonucleotideor using a ribonuclease (RNAse). Therefore, the invention alsoencompasses production of sequencing templates by cleavage of one strand(of a double-stranded nucleic acid molecule) at a site containing one ormore consecutive ribonucleotides using such a chemical cleavage agent oran RNase. Preferably the strand to be cleaved contains a singleribonucleotide to provide a pre-determined site for chemical cleavage.

Suitable chemical cleavage agents capable of selectively cleaving thephosphodiester bond between a deoxyribonucleotide and a ribonucleotideinclude metal ions, for example rare-earth metal ions (especially La³⁺,particularly Tm³⁺, Yb³⁺ or Lu³⁺ (Chen et al. Biotechniques. 2002, 32:518-520; Komiyama et al. Chem. Commun. 1999, 1443-1451)), Fe(3) orCu(3), or exposure to elevated pH, e.g. treatment with a base such assodium hydroxide. By “selective cleavage of the phosphodiester bondbetween a deoxyribonucleotide and a ribonucleotide” is meant that thechemical cleavage agent is not capable of cleaving the phosphodiesterbond between two deoxyribonucleotides under the same conditions.

The base composition of the ribonucleotide(s) is generally not material,but can be selected in order to optimise chemical (or enzymatic)cleavage. By way of example, rUMP or rCMP are generally preferred ifcleavage is to be carried out by exposure to metal ions, especially rareearth metal ions.

The ribonucleotide(s) will typically be incorporated into one strand ofthe double-stranded nucleic acid molecule, and may be situated in aregion thereof which is single-stranded when the two complementarystrands of the double-stranded molecule are annealed (i.e. in a 5′overhanging portion). In particular, if the double-stranded nucleic acidmolecule is prepared by solid-phase PCR amplification using forward andreverse amplification primers, one of which contains at least oneribonucleotide, the standard DNA polymerase enzymes used for PCRamplification are not capable of copying ribonucleotide templates.Hence, the products of the solid-phase PCR reaction will contain anoverhanging 5′ region comprising the ribonucleotide(s) and any remainderof the amplification primer upstream of the ribonucleotide(s).

The phosphodiester bond between a ribonucleotide and adeoxyribonucleotide, or between two ribonucleotides can also be cleavedby an RNase. Any endocytic ribonuclease of appropriate substratespecificity can be used for this purpose. If the ribonucleotide(s) arepresent in a region which is single-stranded when the two complementarystrands of the double-stranded molecule are annealed (i.e. in a 5′overhanging portion), then the RNase will be an endonuclease which hasspecificity for single strands containing ribonucleotides. For cleavagewith ribonuclease it is preferred to include two or more consecutiveribonucleotides, and preferably from 2 to 10 or from 5 to 10 consecutiveribonucleotides. The precise sequence of the ribonucleotides isgenerally not material, except that certain RNases have specificity forcleavage after certain residues. Suitable RNases include, for example,RNaseA, which cleaves after C and U residues. Hence, when cleaving withRNaseA the cleavage site must include at least one ribonucleotide whichis C or U.

Polynucleotides incorporating one or more ribonucleotides can be readilysynthesised using standard techniques for oligonucleotide chemicalsynthesis with appropriate ribonucleotide precursors. If thedouble-stranded nucleic acid molecule is prepared by solid-phase nucleicacid amplification, then it is convenient to incorporate one or moreribonucleotides into one of the primers to be used for the amplificationreaction.

iv) Photochemical Cleavage

The term “photochemical cleavage” encompasses any method which utiliseslight energy in order to achieve cleavage of one or both strands of thedouble-stranded nucleic acid molecule.

A pre-determined site for photochemical cleavage can be provided by anon-nucleotide chemical spacer unit in one of the strands of thedouble-stranded molecule. Suitable photochemical cleavable spacersinclude the PC spacer phosphoamidite(4-(4,4′-Dimethoxytrityloxy)butyramidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite)supplied by Glen Research, Sterling, Va., USA (cat number 10-4913-XX)which has the structure:

The spacer unit can be cleaved by exposure to a UV light source.

This spacer unit can be attached to the 5′ end of a polynucleotide,together with a thiophosphate group which permits attachment to a solidsurface, using standard techniques for chemical synthesis ofoligonucleotides. Conveniently, this spacer unit can be incorporatedinto a forward or reverse amplification primer to be used for synthesisof a photocleavable double-stranded nucleic acid molecule by solid-phaseamplification.

v) Cleavage of Hemimethylated DNA

Site-specific cleavage of one strand of a double-stranded nucleic acidmolecule may also be achieved by incorporating one or more methylatednucleotides into this strand and then cleaving with an endonucleaseenzyme specific for a recognition sequence including the methylatednucleotide(s).

The methylated nucleotide(s) will typically be incorporated in a regionof one strand of the double-stranded nucleic acid molecule having acomplementary stretch of non-methylated deoxyribonucleotides on thecomplementary strand, such that annealing of the two strands produces ahemimethylated duplex structure. The hemimethylated duplex may then becleaved by the action of a suitable endonuclease. For the avoidance ofdoubt, enzymes which cleave such hemimethylated target sequences are notto be considered as “restriction endonucleases” excluded from the scopeof the second aspect of the invention, but rather are intended to formpart of the subject-matter of the invention.

Polynucleotides incorporating one or methylated nucleotides may beprepared using standard techniques for automated DNA synthesis, usingappropriately methylated nucleotide precursors. If the double-strandednucleic acid molecule is prepared by solid-phase nucleic acidamplification, then it is convenient to incorporate one or moremethylated nucleotides into one of the primers to be used for theamplification reaction.

vi) PCR Stoppers

In another embodiment of the invention the double-stranded nucleic acidmay be prepared by solid-phase amplification using forward and reverseprimers, one of which contains a “PCR stopper”. A “PCR stopper” is anymoiety (nucleotide or non-nucleotide) which prevents read-through of thepolymerase used for amplification, such that it cannot copy beyond thatpoint. The result is that amplified strands derived by extension of theprimer containing the PCR stopper will contain a 5′ overhanging portion.This 5′ overhang (other than the PCR stopper itself) may be comprised ofnaturally occurring deoxyribonucleotides, with predominantly naturalbackbone linkages, i.e. it may simply be a stretch of single-strandedDNA. The molecule may then be cleaved in the 5′ overhanging region withthe use of a cleavage reagent (e.g. an enzyme) which is selective forcleavage of single-stranded DNA but not double stranded DNA, for examplemung bean nuclease.

The PCR stopper may be essentially any moiety which preventsread-through of the polymerase to be used for the amplificationreaction. Suitable PCR stoppers include, but are not limited to,hexaethylene glycol (HEG), abasic sites, and any non-natural or modifiednucleotide which prevents read-through of the polymerase, including DNAanalogues such as peptide nucleic acid (PNA).

Stable abasic sites can be introduced during chemical oligonucleotidesynthesis using appropriate spacer units containing the stable abasicsite. By way of example, abasic furan(5′-O-Dimethoxytrityl-1′,2′-Dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)spacers commercially available from Glen Research, Sterling, Va., USA,can be incorporated during chemical oligonucleotide synthesis in orderto introduce an abasic site. Such a site can thus readily be introducedinto an oligonucleotide primer to be used in solid-phase amplification.If an abasic site is incorporated into either forward or reverseamplification primer the resulting amplification product will have a 5′overhang on one strand which will include the abasic site (insingle-stranded form). The single-stranded abasic site may then becleaved by the action of a suitable chemical agent (e.g. exposure toalkali) or an enzyme (e.g. AP-endonuclease VI, Shida et al. NucleicAcids Research, 1996, Vol. 24, 4572-4576).

vii) Cleavage of Peptide Linker

A cleavage site can also be introduced into one strand of thedouble-stranded nucleic molecule by preparing a conjugate structure inwhich a peptide molecule is linked to one strand of the nucleic acidmolecule. The peptide molecule can subsequently be cleaved by apeptidase enzyme of the appropriate specificity, or any other suitablemeans of non-enzymatic chemical or photochemical cleavage. Typically,the conjugate between peptide and nucleic acid will be formed bycovalently linking a peptide to one strand only of the double-strandednucleic acid molecule, with the peptide portion being conjugated to the5′ end of this strand, adjacent to the point of attachment to the solidsurface. If the double-stranded nucleic acid is prepared by solid-phaseamplification, the peptide conjugate may be incorporated at the 5′ endof one of the amplification primers. Obviously the peptide component ofthis primer will not be copied during PCR amplification, hence the“bridged” amplification product will include a cleavable 5′ peptide“overhang” on one strand.

Conjugates between peptides and nucleic acids wherein the peptide isconjugated to the 5′ end of the nucleic acid can be prepared usingtechniques generally known in the art. In one such technique the peptideand nucleic acid components of the desired amino acid and nucleotidesequence can be synthesised separately, e.g. by standard automatedchemical synthesis techniques, and then conjugated in aqueous/organicsolution. By way of example, the OPeC™ system commercially availablefrom Glen Research is based on the “native ligation” of an N-terminalthioester-functionalized peptide to a 5′-cysteinyl oligonucleotide.Pentafluorophenyl S-benzylthiosuccinate is used in the final couplingstep in standard Fmoc-based solid-phase peptide assembly. Deprotectionwith trifluoroacetic acid generates, in solution, peptides substitutedwith an N-terminal S-benzylthiosuccinyl group.O-trans-4-(N-a-Fmoc-5-tert-butylsulfenyl-l-cysteinyl)aminocyclohexyl0-2-cyanoethyl-N,N-diisopropylphosphoramidite is used in the finalcoupling step in standard phosphoramidite solid-phase oligonucleotideassembly. Deprotection with aqueous ammonia solution generates insolution 5′-S-tert-butylsulfenyl-L-cysteinyl functionalizedoligonucleotides. The thiobenzyl terminus of the Modified Peptide isconverted to the thiophenyl analogue by the use of thiophenol, whilstthe Modified Oligonucleotide is reduced using thetris(carboxyethyl)phosphine. Coupling of these two intermediates,followed by the “native ligation” step, leads to formation of theOligonucleotide-Peptide Conjugate.

The conjugate strand containing peptide and nucleic acid can becovalently attached to a solid support using any suitable covalentlinkage technique known in the art which is compatible with the chosensurface. For example, covalent attachment to a solid supportedpolyacrylamide hydrogel surface can be achieved by inclusion of athiophosphate group on the “free” end of the peptide component (i.e. theend not conjugated to the nucleic acid). If the peptide/nucleic acidconjugate structure is an amplification primer to be used forsolid-phase PCR amplification, attachment to the solid support mustleave the 3′ end of the nucleic acid component free.

The peptide component can be designed to be cleavable by any chosenpeptidase enzyme, of which many are known in the art. The nature of thepeptidase is not particularly limited, it is necessary only for thepeptidase to cleave somewhere in the peptide component. Similarly, thelength and amino acid sequence of the peptide component is notparticularly limited except by the need to be “cleavable” by the chosenpeptidase.

The length and precise sequence of the nucleic acid component is alsonot particularly limited, it may be of any desired sequence. If thenucleic acid component is to function as a primer in solid-phase PCR,then its length and nucleotide sequence will be selected to enableannealing to the template to be amplified.

Denaturation

In all embodiments of the invention, regardless of the method used forcleavage, the product of the cleavage reaction may be subjected todenaturing conditions in order to remove the portion(s) of the cleavedstrand(s) that are not attached to the solid support. Suitabledenaturing conditions will be apparent to the skilled reader withreference to standard molecular biology protocols (Sambrook et al.,2001, Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring HarborLaboratory Press, Cold Spring Harbor Laboratory Press, NY; CurrentProtocols, eds Ausubel et al.).

Denaturation (and subsequent re-annealing of the cleaved strands)results in the production of a sequencing template which is partially orsubstantially single-stranded. A sequencing reaction may then beinitiated by hybridisation of a sequencing primer to the single-strandedportion of the template.

In other embodiments of the invention, sequencing can be initiateddirectly after the cleavage step with no need for denaturation to removea portion of the cleaved strand(s). If the cleavage step generates afree 3′ hydroxyl group on one cleaved strand still hybridised to acomplementary strand then sequencing can proceed from this point using astrand-displacement polymerase enzyme without the need for an initialdenaturation step. In particular, strand displacement sequencing may beused in conjunction with template generation by cleavage with nickingendonucleases, or by hydrolysis of an abasic site with endonuclease,heat or alkali treatment.

Features of Solid Supports

In all embodiments of the invention, the term “solid support”, as usedherein, refers to the material to which the polynucleotides moleculesare attached. Suitable solid supports are available commercially, andwill be apparent to the skilled person. The supports can be manufacturedfrom materials such as glass, ceramics, silica and silicon. Supportswith a gold surface may also be used. The supports usually comprise aflat (planar) surface, or at least a structure in which thepolynucleotides to be interrogated are in approximately the same plane.Alternatively, the solid support can be non-planar, e.g., a microbead.Any suitable size may be used. For example, the supports might be on theorder of 1-10 cm in each direction.

The first aspect of the invention relates to generation of sequencingtemplates on a particular type of surface, namely solid supportedpolyacrylamide hydrogels.

As aforesaid, in preparing hydrogel-based solid-supported moleculararrays, a hydrogel is formed and molecules displayed from it. These twofeatures—formation of the hydrogel and construction of the array—may beeffected sequentially or simultaneously.

Where the hydrogel is formed prior to formation of the array, it istypically produced by allowing a mixture of comonomers to polymerise.Generally, the mixture of comonomers contain acrylamide and one or morecomonomers, the latter of which permit, in part, subsequentimmobilisation of molecules of interest so as to form the moleculararray.

The comonomers used to create the hydrogel typically contain afunctionality that serves to participate in crosslinking of the hydrogeland/or immobilise the hydrogel to the solid support and facilitateassociation with the target molecules of interest.

Generally, as is known in the art, polyacrylamide hydrogels are producedas thin sheets upon polymerisation of aqueous solutions of acrylamidesolution. A multiply unsaturated (polyunsaturated) crosslinking agent(such as bisacrylamide) is generally present; the ratio of acrylamide tobisacrylamide is generally about 19:1. Such casting methods are wellknown in the art (see for example Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor Laboratory Press, NY) and need not bediscussed in detail here.

Some form of covalent surface modification of the solid support may bepractised in order to achieve satisfactory immobilisation of eitherhydrogel-based molecular arrays or hydrogels to which it is desired toarray molecules. However, the inventors have observed that suchfunctional modification of the support is not necessary in order toachieve satisfactory immobilisation of arrays of polynucleotides. Inorder to make useful supported arrays capable of binding molecules ofinterest, a mixture of comonomers comprising at least one hydrophilicmonomer and a functionalised comonomer (functionalised to the extentthat the monomer once incorporated into the polymer is capable ofbinding the molecule of interest to the surface of the hydrogel) may bepolymerised so as to form a hydrogel capable of being immobilised on asolid supported, preferably a silica-based, substrate. In particular,the hydrogel may be substantially free of any binder silane components.

In one embodiment the hydrogel may be formed by a method comprisingpolymerising on said support a mixture of:

(i) a first comonomer which is acrylamide, methacrylamide, hydroxyethylmethacrylate or N-vinyl pyrrolidinone; and

(ii) a second comonomer which is a functionalised acrylamide or acrylateof formula (I):

H₂C═C(H)—C(═O)-A-B-C  (I);

or a methacrylate or methacrylamide of formula (II):

or H₂C═C(CH₃)—C(═O)-A-B-C-  (II)

(wherein:

A is NR or O, wherein R is hydrogen or an optionally substitutedsaturated hydrocarbyl group comprising 1 to 5 carbon atoms;

-B- is an optionally substituted alkylene biradical of formula—(CH₂)_(n)— wherein n is an integer from 1 to 50; and wherein n=2 ormore, one or more optionally substituted ethylene biradicals —CH₂CH₂— ofsaid alkylene biradical may be independently replaced by ethenylene andethynylene moieties; and wherein n=1 or more, one or more methylenebiradicals —CH₂— may be replaced independently with an optionallysubstituted mono- or polycyclic hydrocarbon biradical comprising from 4to 50 carbon atoms, or a corresponding heteromonocyclic orheteropolycyclic biradical wherein at least 1 CH₂ or CH₂ is substitutedby an oxygen sulfur or nitrogen atom or an NH group; and

C is a group for reaction with a compound to bind said compoundcovalently to said hydrogel) to form a polymerised product,

characterised in that said method is conducted on, and immobilises thepolymerised product to, said support which is not covalentlysurface-modified.

It has been found that omission of a covalent surface-modification step(particularly of the solid support) affords a surface having greaterpassivity than in the prior art, particularly when compared to thoseinstances where the use of the silane-modifying agents described abovewith silica-based substrates are employed.

The solid upon which the hydrogel is supported is not limited to aparticular matrix or substrate. Suitable supports include silica-basedsubstrates, such as glass, fused silica and other silica-containingmaterials; they may also be silicone hydrides or plastic materials suchas polyethylene, polystyrene, poly(vinyl chloride), polypropylene,nylons, polyesters, polycarbonates and poly(methyl methacrylate).Preferred plastics material are poly(methyl methacrylate), polystyreneand cyclic olefin polymer substrates. Alternatively, other solidsupports may be used such as gold, titanium dioxide, or siliconsupports. The foregoing lists are intended to be illustrative of, butnot limited to, the invention. Preferably, the support is a silica-basedmaterial or plastics material such as discussed herein.

The methods by which the mixture of comonomers are polymerised in theinvention are not characteristic of this invention and will be known tothe skilled person (e.g. by recourse to Sambrook et al. (supra).Generally, however, the polymerisation will be conducted in an aqueousmedium, and polymerisation initiated by any suitable initiator.Potassium or ammonium persulfate as an initiator is typically employed.Tetramethylethylenediamine (TMEDA or TEMED) may be and generally is usedto accelerate the polymerisation.

It is not necessary that, a polyunsaturated crosslinking agent such asbisacrylamide or pentaerythritol tetraacrylate is present in the mixturewhich is polymerised; nor is it necessary to form PRP-type intermediatesand crosslink them.

Generally, in producing hydrogels according to this invention, only onecompound of formulae (I) or (II) will be used. Use of a compound of theformulae (I) or (II) permits formation of a hydrogel capable of beingimmobilised to solid supports, preferably silica-based solid supports.The compounds of these formulae comprise portions A, B and C as definedherein.

Biradical A may be oxygen or N(R) wherein R is hydrogen or a C₁₋₅ alkylgroup. Preferably, R is hydrogen or methyl, particularly hydrogen. WhereR is a C₁₋₅ alkyl group, this may contain one or more, e.g. one to threesubstituents. Preferably, however, the alkyl group is unsubstituted.

Biradical B is a predominantly hydrophobic linking moiety, connecting Ato C and may be an alkylene biradical of formula —(CH₂)_(n)—, wherein nis from 1 to 50. Preferably n is 2 or more, e.g. 3 or more. Preferably nis 2 to 25, particularly 2 to 15, more particularly 4 to 12, for example5 to 10.

Where n in —(CH₂)_(n)— is 2 or more, one or more biradicals —CH₂CH₂—(-ethylene-) may be replaced with ethenylene or ethynylene biradicals.Preferably, however, the biradical B does not contain such unsaturation.

Additionally, or alternatively, where n in —(CH₂)_(n)— is 1 or more, oneor more methylene radicals —CH₂— in B may be replaced with a mono- orpolycyclic biradical which preferably comprises 5 to 10 carbon atomse.g. 5 or 6 carbon atoms. Such cyclic biradicals may be, for example,1,4-, 1,3- or 1,2-cyclohexyl biradicals. Bicylic radicals such asnapthyl or decahydronaphthyl may also be employed. Correspondingheteroatom-substituted cyclic biradicals to those homocyclic biradicalsmay also be employed, for example pyridyl, piperidinyl, quinolinyl anddecahydroquinolinyl.

It will be appreciated that the scope of -B- is thus not particularlyrestricted. Most preferably, however, -B- is a simple, unsubstituted,unsaturated alkylene biradical such as a C₃-C₁₀ alkylene group,optimally C₅-C₈, such as n-pentylene: —(CH₂)₅—.

Where an alkyl group (or alkylene, alkenylene etc) is indicated as being(optionally) substituted, substituents may be selected from the groupcomprising hydroxyl, halo (i.e. bromo, chloro, fluoro or iodo),carboxyl, aldehydro, amine and the like. The biradical -B- is preferablyunsubstituted or substituted by fewer than 10, preferably fewer than 5,e.g. by 1, 2 or 3 such substituents.

Group C serves to permit attachment of molecules of interest afterformation of the hydrogel. The nature of Group C is thus essentiallyunlimited provided that it contains a functionality allowing reactionbetween the hydrogel and the molecules to be immobilised. Preferably,such a functionality will not require modification prior to reactionwith the molecule of interest and thus the C group is ready for directreaction upon formation of the hydrogel. Preferably such a functionalityis a hydroxyl, thiol, amine, acid (e.g. carboxylic acid), ester andhaloacetamido, haloacetamido and in particular bromoacetamido beingparticularly preferred. Other appropriate C groups will be evident tothose skilled in the art and include groups comprising a singlecarbon-carbon double bond which is either terminal (i.e. where a C grouphas an extremity terminating in a carbon-carbon double bond) or wherethe carbon-carbon double bond is not at a terminal extremity. When a Cgroup comprises a carbon-carbon double bond, this is preferably fullysubstituted with C₁₋₅ alkyl groups, preferably methyl or ethyl groups,so that neither carbon atom of the C═C moiety bears a hydrogen atom.

The C moiety may thus comprise, for example, a dimethylmaleimide moietyas disclosed in U.S. Pat. No. 6,372,813, WO01/01143, WO02/12566 andWO03/014394.

The (meth)acrylamide or (meth)acrylate of formula (I) or (II) which iscopolymerised with acrylamide, methacrylamide, hydroxyethyl methacrylateor N-vinyl pyrrolidinone is preferably an acrylamide or acrylate, i.e.of formula (I). More preferably it is an acrylamide and still morepreferably it is an acrylamide in which A is NH.

The reaction between a comonomer of formula (I) or (II) and acrylamide,methacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinonemethacrylamide, particularly acrylamide, has been found to affordhydrogels particularly suitable for use in the generation of moleculararrays. However, it will be appreciated by those skilled in the art thatanalogous copolymers may be formed by the reaction between comonomers offormula (I) or (II) and any vinylogous comonomer,hydroxyethylmethacrylate and n-vinyl pyrrolidinone being two examples ofsuch vinylogous comonomers.

Control of the proportion of monomer of formula (I) or (II) to that ofthe first comonomer (e.g. acrylamide and/or methacrylamide, preferablyacrylamide) allows adjustment of the physical properties of the hydrogelobtained on polymerisation. It is preferred that the comonomer offormula (I) or (II) is present in an amount of 1 mol %, preferably ≧2mol % (relative to the total molar quantity of comonomers) in order forthe hydrogel to have optimum thermal and chemical stability underconditions typically encountered during the preparation, and subsequentmanipulation, of the molecular arrays produced from the hydrogels.Preferably, the amount of comonomer of formula (I) or (II) is less thanor equal to about 5 mol %, more preferably less than or equal to about 4mol %. Typical amounts of comonomer of formula (I) or (II) used are1.5-3.5 mol %, exemplified herein by about 2% and about 3%.

The amounts of acrylamide or methacrylamide from which the hydrogels areprimarily constructed are those typically used to form hydrogels, e.g.about 1 to about 10% w/v, preferably less than 5 or 6% w/v, e.g. about 1to about 2% w/v. Again, of course, the precise nature of the hydrogelmay be adjusted by, in part, control of the amount of acrylamide ormethacrylamide used.

When forming the hydrogels, acrylamide or methacrylamide may bedissolved in water and mixed with a solution of a comonomer of formula(I) or (II). The latter may be conveniently dissolved in awater-miscible solvent, such as dimethylformamide (DMF), or wateritself. The most appropriate solvent may be selected by the skilledperson and shall depend upon the structure of the comonomer of formula(I) or (II).

The methods by which the monomers of formula (I) or (II) are synthesisedwill be evident to those skilled in the art. By way of example, thesynthesis of a particularly preferred monomer (of formula (I) whereinA=NH, -B-=—(CH₂)₅— and —C═—N(H)—C(═O)CH₂Br is provided as an examplehereinafter.

As noted above, the general methods by which the polymerisation iscarried out are known to those skilled in the art. For example,generally acrylamide or methacrylamide is dissolved in purified water(e.g. Milli Q) and potassium or ammonium persulfate dissolved separatelyin purified water. The comonomer of formula (I) or (II) may beconveniently dissolved in a water-miscible organic solvent, e.g.glycerol, ethanol, methanol, dimethylformamide (DMF) etc. TEMED may beadded as appropriate. Once formulated (a typical preparation isdescribed in the examples), the mixture is polymerised with as littledelay as possible after its formulation.

The polymerisation process may be conducted by any convenient means.

The second aspect of the invention is not limited with respect to thenature of the solid support and can be used in conjunction withessentially any type of support known in the art for production ofpolynucleotide arrays, and more specifically with any type of supportwhich is compatible with solid-phase nucleic acid amplification.Suitable supports include those coated with functionalised silanes. Inparticular, the method of the second aspect of the invention can be usedin conjunction with the silane-coated solid supports described in WO98/44151 and WO 00/18957.

Construction of Arrays by Solid Phase Amplification

In a particular embodiment of the invention, the methods according tothe first and second aspects of the invention may be used to preparetemplates for sequencing starting from double-stranded nucleic acidmolecules present in clustered arrays of nucleic acid colonies generatedby solid-phase nucleic acid amplification. In this context, the term“solid-phase amplification” refers to an amplification reaction which isanalogous to standard PCR, except that the forward and reverseamplification primers are immobilised (e.g. covalently attached) to asolid support at or near the 5′ end. The products of the PCR reactionare thus extended strands derived by extension of the amplificationprimers that are immobilised on the solid support at or near the 5′ end.Solid-phase amplification may itself be carried out, for example, usingprocedures analogous to those described in WO 98/44151 and WO 00/18957,the contents of which are incorporated herein in their entirety byreference.

As a first step in colony generation by solid-phase amplification amixture of forward and reverse amplification primers may be immobilisedor “grafted” onto the surface of a suitable solid support. The graftingstep will generally involve covalent attachment of the primers to thesupport at or near the 5′ end, leaving the 3′ end free for primerextension.

The amplification primers are typically oligonucleotide molecules havethe following structures:

Forward primer: A-L-S1Reverse primer: A-L-S2

Wherein A represents a moiety which allows attachment to a solidsupport, L represents an optional linker moiety and S1 and S2 arepolynucleotide sequences which permit amplification of a substratenucleic acid molecule comprising a target region that it is desired to(fully or partially) sequence.

The mixture of primers grafted onto the solid support will generallycomprise substantially equal amounts of the forward and reverse primers.

Group A can be any moiety (including a non-nucleotide chemicalmodification) which enables attachment (preferably covalent) to a solidsupport. In all aspects of the invention group A may comprise asulphur-containing nucleophile, such as phosphorothioate, present at the5′ end of a polynucleotide strand. The most preferred means of graftingprimers to a solid support is via 5′ phosphorothioate attachment to ahydrogel comprised of polymerised acrylamide andN-(5-bromoacetamidylpentyl)acrylamide (BRAPA).

L represents a linker or spacer which may be included but is notstrictly necessary. The linker may be included in order to ensure that acleavage site present in the immobilised polynucleotide moleculesgenerated as a result of the amplification reaction is positioned at anoptimum distance from the solid support, or the linker may itselfcontain a cleavage site.

The linker may be a carbon-containing chain such as those of formula(CH₂)_(n) wherein “n” is from 0.1 to about 1500, for example less thanabout 1000, preferably less than 100, e.g. from 2-50, particularly 5-25.However, a variety of other linkers may be employed with the onlyrestriction placed on their structures being that the linkers are stableunder conditions under which the polynucleotides are intended to be usedsubsequently, e.g. conditions used in DNA amplification, cleavage andsequencing.

Linkers which do not consist of only carbon atoms may also be used. Suchlinkers include polyethylene glycol (PEG) having a general formula of(CH₂—CH₂—O)_(m), wherein m is from about 1 to 600, preferably less thanabout 500.

Linkers formed primarily from chains of carbon atoms and from PEG may bemodified so as to contain functional groups which interrupt the chains.Examples of such groups include ketones, esters, amines, amides, ethers,thioethers, sulfoxides, sulfones. Separately or in combination with thepresence of such functional groups may be employed alkene, alkyne,aromatic or heteroaromatic moieties, or cyclic aliphatic moieties (e.g.cyclohexyl). Cyclohexyl or phenyl rings may, for example, be connectedto a PEG or (CH₂)_(n) chain through their 1- and 4-positions.

As an alternative to the linkers described above, which are primarilybased on linear chains of saturated carbon atoms, optionally interruptedwith unsaturated carbon atoms or heteroatoms, other linkers may beenvisaged which are based on nucleic acids or monosaccharide units (e.g.dextrose). It is also within the scope of this invention to utilisepeptides as linkers.

In a further embodiment the linker may comprise one or more nucleotides.Such nucleotides may also be referred to herein as “spacer” nucleotides.Typically from 1 to 20, more preferably from 1 to 15 or from 1 to 10,and more particularly 2, 3, 4, 5, 6, 7, 8, 9 or 10 spacer nucleotidesmay be included. Most preferably the primer will include 10 spacernucleotides. It is preferred to use polyT spacers, although othernucleotides and combinations thereof can be used. In one preferredembodiment the primer may include 10T spacer nucleotides.

For the primer grafting reaction to proceed a mixture of theamplification primers is applied to a solid support under conditionswhich permit reaction between moiety A and the support. The solidsupport may be suitably functionalised to permit covalent attachment viamoiety A. The result of the grafting reaction is a substantially evendistribution of the primers over the solid support.

In certain embodiments the template to be amplified may be grafted ontothe solid support together with the amplification primers in a singlegrafting reaction. This can be achieved by adding template moleculesincluding moiety A at the 5′ end to the mixture of primers to form aprimer-template mixture. This mixture is then grafted onto the solidsupport in a single step. Amplification may then proceed using theimmobilised template and primers in a reaction analogous to thatdescribed in WO 00/18957. The first step in such a reaction will behybridisation between surface-bound templates and surface-boundamplification primers.

If a mixture of primers only is grafted onto the solid support, thetemplate to be amplified may be added in free solution. Theamplification reaction may then proceed substantially as described in WO98/44151. Briefly, following attachment of the primers the solid supportis contacted with the template to be amplified under conditions whichpermit hybridisation between the template and the immobilised primers.The template is usually added in free solution under suitablehybridisation conditions, which will be apparent to the skilled reader.Typically hybridisation conditions are, for example, 5×SSC at 40° C.,following an initial denaturation step. Solid-phase amplification canthen proceed, the first step of the amplification being a primerextension step in which nucleotides are added to the 3′ end of theimmobilised primer hybridised to the template to produce a fullyextended complementary strand. This complementary strand will thusinclude at its 3′ end a sequence which is capable of binding to thesecond primer molecule immobilised on the solid support. Further roundsof amplification (analogous to a standard PCR reaction) lead to theformation of clusters or colonies of template molecules bound to thesolid support.

Sequences S1 and S2 in the amplification primers may be specific for aparticular target nucleic acid that it is desired to amplify, but inother embodiments sequences S1 and S2 may be “universal” primersequences which enable amplification of any target nucleic acid of knownor unknown sequence which has been modified to enable amplification withthe universal primers.

Suitable substrates to be amplified with universal primers may beprepared by modifying polynucleotides comprising the target region to beamplified (and sequenced) by addition of known adaptor sequences to the5′ and 3′ ends of the target polynucleotides to be amplified. The targetmolecules themselves may be any double-stranded polynucleotide moleculesit is desired to sequence (e.g. random fragments of human genomic DNA).The adaptor sequences enable amplification of these molecules on a solidsupport to form clusters using forward and reverse primers having thegeneral structure described above, wherein sequences S1 and S2 areuniversal primer sequences.

The adaptors are typically short oligonucleotides that may besynthesised by conventional means. The adaptors may be attached to the5′ and 3′ ends of target nucleic acid fragments by a variety of means(e.g. subcloning, ligation. etc). More specifically, two differentadaptor sequences are attached to a target nucleic acid molecule to beamplified such that one adaptor is attached at one end of the targetnucleic acid molecule and another adaptor is attached at the other endof the target nucleic acid molecule. The resultant construct comprisinga target nucleic acid sequence flanked by adaptors may be referred toherein as a “substrate nucleic acid construct”. The targetpolynucleotides may advantageously be size-fractionated prior tomodification with the adaptor sequences.

The adaptors contain sequences which permit nucleic acid amplificationusing the amplification primer molecules immobilised on the solidsupport. These sequences in the adaptors may be referred to herein as“primer binding sequences”. In order to act as a template for nucleicacid amplification, a single strand of the template construct mustcontain a sequence which is complementary to sequence S1 in the forwardamplification primers (such that the forward primer molecule can bindand prime synthesis of a complementary strand) and a sequence whichcorresponds to sequence S2 in the reverse amplification primer molecules(such that the reverse primer molecule can bind to the complementarystrand). The sequences in the adaptors which permit hybridisation toprimer molecules will typically be around 20-40 nucleotides in length,although the invention is not limited to sequences of this length.

The precise identity of sequences S1 and S2 in the amplificationprimers, and hence the cognate sequences in the adaptors, are generallynot material to the invention, as long as the primer molecules are ableto interact with the amplification sequences in order to direct PCRamplification. The criteria for design of PCR primers are generally wellknown to those of ordinary skill in the art.

Solid-phase amplification by either the method analogous to that of WO98/44151 or that of WO 00/18957 will result in production of a clusteredarray comprised of colonies of “bridged” amplification products. Bothstrands of the amplification products will be immobilised on the solidsupport at or near the 5′ end, this attachment being derived from theoriginal attachment of the amplification primers. Typically theamplification products within each colony will be derived fromamplification of a single template (target) molecule.

Modifications required to enable subsequent cleavage of the bridgedamplification products may be advantageously included in one or bothamplification primers. Such modifications may be placed anywhere in theamplification primer, provided this does not affect the efficiency ofthe amplification reaction to a material extent. Thus, the modificationswhich enable cleavage may form part of the linker region L or one orboth of sequences S1 or S2. By way of example, the amplification primersmay be modified to include inter alia diol linkages, uracil nucleotides,ribonucleotides, methylated nucleotides, peptide linkers, PCR stoppersor recognition sequences for a restriction endonuclease. Because allnucleic acid molecules prepared by solid-phase amplification willultimately contain sequences derived from the amplification primers, anymodifications in the primers will be carried over into the amplifiedproducts.

Therefore, in a further aspect the invention provides a method ofsolid-phase nucleic acid amplification wherein forward and reverseamplification primers covalently attached to a solid support at the 5′end are used to amplify one or more polynucleotide templates, the methodbeing characterised in that one or both of the amplification primersincludes a site for cleavage by a method other than digestion with therestriction endonuclease or a nicking endonuclease.

Preferred features of the amplification primers are as described aboveand preferred cleavage methods are as described above in connection withthe first and second aspects of the invention. Other preferred featuresdescribed in relation to the first and second aspects of the inventionapply mutatis mutandis to this aspect of the invention.

The utility of the “template formation” method of the first and secondaspects of the invention is not limited to formation of templates fromdouble-stranded nucleic acids produced by an amplification reaction,although this is preferred. The method may be applied to linearisationof immobilised double-stranded polynucleotides produced on a support byany other means.

Methods of Sequencing

The invention also encompasses method of sequencing nucleic acidtemplates generated using the methods of the invention. Thus, theinvention provides a method of nucleic acid sequencing comprisingforming a template for nucleic acid sequencing using a method accordingto the first or second aspect of the invention and performing a nucleicacid sequencing reaction to determine the sequence one at least oneregion of the template.

Sequencing can be carried out using any suitable“sequencing-by-synthesis” technique, wherein nucleotides are addedsuccessively to a free 3′ hydroxyl group, resulting in synthesis of apolynucleotide chain in the 5′ to 3′ direction. The nature of thenucleotide added is preferably determined after each addition.

An initiation point for a sequencing reaction may be provided byannealing of a sequencing primer to a single-stranded region of thetemplate. Thus, the invention encompasses methods wherein the nucleicacid sequencing reaction comprises hybridising a sequencing primer to asingle-stranded region of the template generated in step (iii) of the“template generation” method of the first or second aspect of theinvention, sequentially incorporating one or more nucleotides into apolynucleotide strand complementary to the region of the template to besequenced, identifying the base present in one or more of theincorporated nucleotide(s) and thereby determining the sequence of aregion of the template.

In other embodiments of the invention the initiation point for thesequencing reaction may be a free 3′ hydroxyl group generated bycleavage of one strand of the template itself (e.g. by cleavage of anabasic nucleotide with endonuclease, heat or alkali). Thus, theinvention also provides methods of sequencing which involve forming atemplate for nucleic acid sequencing using a method comprising steps (i)and (ii) only of the “template formation” methods of the first or secondaspects of the invention, carrying out a strand displacement sequencingreaction by sequentially adding one or more nucleotides to a free 3′hydroxyl group generated on the strand cleaved in step identifying thebase present in one or more of the incorporated nucleotide(s) andthereby determining the sequence of a region of the template.

A preferred sequencing method which can be used in all aspects of theinvention relies on the use of modified nucleotides containing 3′blocking groups that can act as chain terminators. Once the modifiednucleotide has been incorporated into the growing polynucleotide chaincomplementary to the region of the template being sequenced there is nofree 3′-OH group available to direct further sequence extension andtherefore the polymerase can not add further nucleotides. Once thenature of the base incorporated into the growing chain has beendetermined, the 3′ block may be removed to allow addition of the nextsuccessive nucleotide. By ordering the products derived using thesemodified nucleotides it is possible to deduce the DNA sequence of theDNA template. Such reactions can be done in a single experiment if eachof the modified nucleotides has attached a different label, known tocorrespond to the particular base, to facilitate discrimination betweenthe bases added at each incorporation step. Alternatively, a separatereaction may be carried out containing each of the modified nucleotidesseparately.

The modified nucleotides may carry a label to facilitate theirdetection. Preferably this is a fluorescent label. Each nucleotide typemay carry a different fluorescent label. However the detectable labelneed not be a fluorescent label. Any label can be used which allows thedetection of an incorporated nucleotide.

One method for detecting fluorescently labelled nucleotides comprisesusing laser light of a wavelength specific for the labelled nucleotides,or the use of other suitable sources of illumination. The fluorescencefrom the label on the nucleotide may be detected by a CCD camera orother suitable detection means.

The methods of the invention are not limited to use of the sequencingmethod outlined above, but can be used in conjunction with essentiallyany sequencing methodology which relies on successive incorporation ofnucleotides into a polynucleotide chain. Suitable techniques include,for example, Pyrosequencing™, FISSEQ (fluorescent in situ sequencing),MPSS (massively parallel signature sequencing) and sequencing byligation-based methods.

The target polynucleotide to be sequenced using the method of theinvention may be any polynucleotide that it is desired to sequence. Thetarget polynucleotide may be of known, unknown or partially knownsequence, for example in re-sequencing applications. Using the templatepreparation method described in detail herein it is possible to preparetemplates starting from essentially any double-stranded targetpolynucleotide of known, unknown or partially known sequence. With theuse of arrays it is possible to sequence multiple targets of the same ordifferent sequence in parallel. A particularly preferred application ofthe method is in the sequencing of fragments of genomic DNA.

The invention will be further understood with reference to the followingnon-limiting experimental examples.

BACKGROUND

The use of diol within DNA or oligonucleotides has been widely reportedin the literature as a cleavable linker or as masked aldehydefunctionality and used for diverse applications as mutagenesis (Karionet al. (2001) Nucl. Acids Res. 29(12), 2456-2463), enzyme mechanismstudy (Brevnov M. G. et al. (1997) Nucl. Acids Res. 25(16), 3302-3309),post-synthesis modifications of oligonucleotides (Ollivier N. et al.(2002) Tet. Lett. 43, 997-999) or new solid support for peptideautomated synthesis (Melnyk O. et al. (2001) J. Org. Chem. 66(12),4153-4160). The deprotection method has been proven compatible with DNAintegrity.

General Methods

The following are general methods used in the accompanying examples

Acrylamide Coating of Glass Chips

The solid supports used in this experiment were 8-channel glass chipssuch as those provided by Micronit (Twente, Nederland) or IMT(Neuchâtel, Switzerland). However, the experimental conditions andprocedures are readily applicable to other solid supports.

Chips were washed as follows: neat Decon for 30 min, milliQ H₂O for 30min, NaOH 1N for 15 min, milliQ H₂O for 30 min, HCl 0.1N for 15 min,milliQ H₂O for 30 min.

Polymer Solution Preparation

For 10 ml of 2% polymerisation mix.

-   -   10 ml of 2% solution of acrylamide in milliQ H2O    -   165 μl of a 100 mg/ml N-(5-bromoacetamidylpentyl)acrylamide        (BRAPA) solution in DMF (23.5 mg in 235 μl DMF)    -   11.5 μl of TEMED    -   100 μl of a 50 mg/ml solution of potassium persulfate in milliQ        H₂O (20 mg in 400 μl H₂O)

The 10 ml solution of acrylamide was first degassed with argon for 15min. The solutions of BRAPA, TEMED and potassium persulfate weresuccessively added to the acrylamide solution. The mixture was thenquickly vortexed and immediately used. Polymerization was then carriedout for 1 h 30 at RT. Afterwards the channels were washed with milliQH₂O for 30 min. The slide was then dried by flushing argon through theinlets and stored under low pressure in a dessicator.

Synthesis of N-(5-bromoacetamidylpentyl)acrylamide (BRAPA)

N-Boc-1,5-diaminopentane toluene sulfonic acid was obtained fromNovabiochem. The bromoacetyl chloride and acryloyl chloride wereobtained from Fluka. All other reagents were Aldrich products.

To a stirred suspension of N-Boc-1,5-diaminopentane toluene sulfonicacid (5.2 g, 13.88 mmol) and triethylamine (4.83 ml, 2.5 eq) in THF (120ml) at 0° C. was added acryloyl chloride (1.13 ml, 1 eq) through apressure equalized dropping funnel over a one hour period. The reactionmixture was then stirred at room temperature and the progress of thereaction checked by TLC (petroleum ether:ethyl acetate 1:1). After twohours, the salts formed during the reaction were filtered off and thefiltrate evaporated to dryness. The residue was purified by flashchromatography (neat petroleum ether followed by a gradient of ethylacetate up to 60%) to yield 2.56 g (9.98 mmol, 71%) of product 2 as abeige solid. ¹H NMR (400 MHz, d₆-DMSO): 1.20-1.22 (m, 2H, CH₂),1.29-1.43 (m, 13H, tBu, 2×CH₂), 2.86 (q, 2H, J=6.8 Hz and 12.9 Hz, CH₂),3.07 (q, 2H, J=6.8 Hz and 12.9 Hz, CH₂), 5.53 (dd, 1H, J=2.3 Hz and 10.1Hz, CH), 6.05 (dd, 1H, J=2.3 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 Hz, CH), 6.77 (t, 1H, J=5.3 Hz, NH), 8.04 (bs, 1H, NH). Mass(electrospray+) calculated for C₁₃H₂₄N₂O₃ 256. found 279 (256+Na⁺).

Product 2 (2.56 g, 10 mmol) was dissolved in trifluoroaceticacid:dichloromethane (1:9, 100 ml) and stirred at room temperature. Theprogress of the reaction was monitored by TLC (dichloromethane:methanol9:1). On completion, the reaction mixture was evaporated to dryness, theresidue co-evaporated three times with toluene and then purified byflash chromatography (neat dichloromethane followed by a gradient ofmethanol up to 20%). Product 3 was obtained as a white powder (2.43 g, 9mmol, 90%). ¹H NMR (400 MHz, D₂O): 1.29-1.40 (m, 2H, CH₂), 1.52 (quint.,2H, J=7.1 Hz, CH₂), 1.61 (quint., 2H, J=7.7 Hz, CH₂), 2.92 (t, 2H, J=7.6Hz, CH₂), 3.21 (t, 2H, J=6.8 Hz, CH₂), 5.68 (dd, 1H, J=1.5 Hz and 10.1Hz, CH), 6.10 (dd, 1H, J=1.5 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 Hz, CH). Mass (electrospray+) calculated for C₈H₁₆N₂O 156.found 179 (156+Na⁺).

To a suspension of product 3 (6.12 g, 22.64 mmol) and triethylamine(6.94 ml, 2.2 eq) in THF (120 ml) was added bromoacetyl chloride (2.07ml, 1.1 eq), through a pressure equalized dropping funnel, over a onehour period and at −60° C. (cardice and isopropanol bath in a dewar).The reaction mixture was then stirred at room temperature overnight andthe completion of the reaction was checked by TLC(dichloromethane:methanol 9:1) the following day. The salts formedduring the reaction were filtered off and the reaction mixtureevaporated to dryness. The residue was purified by chromatography (neatdichloromethane followed by a gradient of methanol up to 5%). 3.2 g(11.55 mmol, 51%) of the product 1 (BRAPA) were obtained as a whitepowder. A further recrystallization performed in petroleum ether:ethylacetate gave 3 g of the product 1. ¹H NMR (400 MHz, d₆-DMSO): 1.21-1.30(m, 2H, CH₂), 1.34-1.48 (m, 4H, 2×CH₂), 3.02-3.12 (m, 4H, 2×CH₂), 3.81(s, 2H, CH₂), 5.56 (d, 1H, J=9.85 Hz, CH), 6.07 (d, 1H, J=16.9 Hz, CH),6.20 (dd, 1H, J=10.1 Hz and 16.9 Hz, CH), 8.07 (bs, 1H, NH), 8.27 (bs,1H, NH). Mass (electrospray+) calculated for C₁₀H₁₇BrN₂O₂ 276 or 278.found 279 (278+H⁺), 299 (276+Na⁺).

Grafting of Primers

The primers were 5′-phosphorothioate oligonucleotides. Their sequencesand suppliers varied according to the experiment they were used for (seespecific examples).

Grafting was carried out using 80 μl per channel in 10 mM phosphatebuffer pH7 for 1 h at RT.

Colony Formation

A PCR template may be hybridised to the grafted primers immediatelyprior to the PCR reaction. The PCR reaction thus begins with an initialprimer extension step rather than template denaturation.

The hybridization procedure begins with a heating step in a stringentbuffer (95° C. for 5 minutes in TE) to ensure complete denaturationprior to hybridisation of the PCR template. Hybridization was thencarried out in 5×SSC, using template diluted to the desired finalconcentration. After the hybridization, the chip was washed for 5minutes with milliQ water to remove salts.

Surface amplification was carried out by thermocycled PCR in an MJResearch thermocycler.

A typical PCR program is as follows:

1—97.5° C. for 0:45 2—X° C. for 1:30 3—73° C. for 1:30

4—Goto 1 [40] times

5—73° C. for 5:00 6—20° C. for 3:00 7—End

Since the first step in the amplification reaction was extension of theprimers bound to template in the initial hybridisation step the firstdenaturation and annealing steps of this program are omitted (i.e. thechip is placed on the heating block only when the PCR mix is pumpedthrough the flow cell and the temperature is at 73° C.).

The annealing temperature (X° C., step 2) depends on the primer pairthat is used. Experiments have determined an optimal annealingtemperature of 57° C. for P5/P7 primers. For other primer-pairs theoptimum annealing temperature can be determined by experiment. Thenumber of PCR cycles may be varied if required.

PCR was carried out in a reaction solution comprising 1×PCR reactionbuffer (supplied with the enzyme) 1M betain, 1.3% DMSO, 200 μM dNTPs and0.025 U/μL Taq polymerase.

General features of the solid-phase amplification procedure to producenucleic acid colonies are as described in International patentapplications WO 98/44151 and WO 00/18957.

Linearisation

See procedures described in the specific examples

Thermal Dehybridisation

Thermal denaturation or de-hybridization of linearised colonies wascarried out in stringent buffer (TE).

Temperature was ramped 0.5° C./sec to 97.5° C. and held at 97.5° C. for2 minutes 30 seconds.

Hybridisation of Sequencing Primer

The procedure begins with a heating step in a stringent buffer (TE) toensure complete denaturation of the colonies prior to hybridisation ofthe primer.

Hybridization was carried out in 5×SSC, using an oligonucleotide dilutedto a final concentration of 500 nM. This solution should be preparedjust before use, especially when fluorophore-labelled oligonucleotidesare used.

Typical temperature cycling profile was as follows:

MJ-Research Thermocycler program set:

-   -   (Control method: block)    -   1—0.5° C./sec to 97.5° C.    -   2—97.5° C. for 2:30    -   3—97.5° C. for 0:02        -   −0.1° C. per cycle    -   4—Goto 3 for 574 times    -   5—40° C. for 15:00    -   6—End

Example 1 Cleavage Evaluation of Diol Primer in 8 Channel (Baccarat)Chip Oligonucleotides Used: Labelled P5 Complementary Oligonucleotide(Supplied by Eurogentec):

(SEQ ID NO: 1) 5′-Texas Red-TCGGTGGTCGCCGTATCATT-3′-OH

Grafting Control Primer (Supplied by Eurogentec):

(SEQ ID NO: 2) 5′-phosphorothioate-GTAGACTGCATGACCTGTAG-3′-Cy3

P5 Non-Cleavable Primer (Supplied by Eurogentec):

(SEQ ID NO: 3) 5′-phosphorothioate-TTTTTTTTTTAATGATACGGCGACCACC GA -3′OH

P5 Cleavable Primer (Supplied by Fidelity Systems):

(SEQ ID NO: 4) 5′-phosphorothioate-arm 26-diol22A-AATGATACGGCGACCACCGA-3′OH

The structures of the arm26 and dio122A components were as follows:

Grafting was performed according to the procedure described undergeneral methods. Channels 1 and 2 of an 8 channel chip were graftedusing the non-cleavable primer, channels 5, 6 and 7 using the cleavablediol linker and channel 8 using the grafting control.

A first hybridization using the complementary P5 oligonucleotide (SEQ IDNO:1) was performed to measure the percentage of P5 oligonucleotidesattached to the surface. Standard hybridisation conditions are describedin the general methods above. Channels to be cleaved were treated with asolution of 0.1M sodium periodate in water for 30 min at roomtemperature, channel 1 being kept as a control to evaluate the potentialdamage of the DNA or the surface by sodium periodate. The channels werethen washed for 5 minutes with milliQ water then 5 min 5×SSC buffer atroom temperature.

A second hybridization was carried out using the complementary P5oligonucleotide (SEQ ID NO:1) to evaluate the percentage of non-cleavedoligonucleotide. A second treatment with 0.1M sodium periodate was thenperformed for 15 min followed by the same washing conditions and a lasthybridization using the complementary P5 oligonucleotide. After eachhybridization, the chip was scanned using the following settings: focalplane +3 mm, 600V, 610BP/Red (633 nm). The intensities were thennormalized.

Results

The results illustrated graphically in FIG. 4 show that the diol linker(SEQ ID NO:4) was cleaved with an efficiency of 70%.

Example 2 Nucleic Acid Colony Formation on Acrylamide (SFA) Coated 8Channel (Baccarat) Chips Using Diol Primer Oligonucleotides Used: P5Non-Cleavable Primer (Supplied by Eurogentec):

(SEQ ID NO: 3) 5′-phosphorothioate-TTTTTTTTTTAATGATACGGCGACCACCG A -3′OH

P7 Non-Cleavable Primer (Supplied by Eurogentec):

(SEQ ID NO: 5) 5′-phosphorothioate- TTTTTTTTTTCAAGCAGAAGACGGCATACGA -3′OH

P5 Cleavable Primer (Supplied by ATD):

5′-phosphorothioate-TTTTTTTTTT-(diol)X3-AATGATACG GCGACCACCGA -3′OH(equivalent to SEQ ID NO:3 but including diol linkage)

The structure of the “diol” linker incorporated into the cleavableprimer was as follows:

It will be noted that the linker unit is incorporated with the diol in aprotected OAc form during oligonucleotide synthesis. The free diol isreleased by ammonia treatment during oligonucleotidecleavage/deprotection. Therefore, the primers used in the graftingreaction contain the free diol.

Grafting was performed according to the procedure described in thegeneral methods. Channels 1, 2 and 3 of an 8 channel chip were graftedusing the non-cleavable pair of P5/P7 primers (SEQ ID NOS 3 and 5),channels 4, 5, 6, 7 and 8 using the cleavable P5 diol primer illustratedabove (equivalent to SEQ ID NO:3 but including diol linkage between thepolyT portion and the P5 portion) with non-cleavable primer P7 (SEQ IDNO:5).

The template used for amplification was a library of PCR fragmentsderived from PhiX 174. The fragments contain common “end” sequencesenabling amplification with the P5 and P7 primers flanking 400-700 bpfragments of PhiX 174 of unknown sequence. Hybridization of the templatewas carried out substantially as described in the general methods aboveusing a 100 pM solution of template for channels 2, 3, 4, 5, 6 and 7,channels 1 and 8 being kept as primer only controls. PCR was performedas described in the general methods. Amplification products were thenstained with SyBr Green-I in TE buffer (1/10 000), using 100 μl perchannel and visualised using objective 0.4, Filter Xf 22 and 1 secondacquisition time (gain 1).

Results

FIGS. 5 and 6 illustrate that successful colony formation was obtainedusing the cleavable diol primer.

Example 3 Cleavage and Subsequent Hybridization of Nucleic Acid ColoniesGenerated on Diol Cleavable Primer Olionucleotide Used:

A594 sequencing primer (supplied by Eurogentec):

(SEQ ID NO: 6) 5′-A594- CTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGT G-3′-OH

Channels to be cleaved were treated with a solution of 0.1M of sodiumperiodate and 0.1M ethanolamine in water for 1 hour at room temperature.All the other channels were washed with milliQ water. All channels werethen washed for 30 minutes with milliQ water at room temperature.

Hybridization was carried out using the sequencing primer (SEQ ID NO:6)labelled with A594 to evaluate the percentage of non-cleavedoligonucleotide. The sequencing primer was hybridised to the linearisedclusters prepared as described above at 500 nM, using standardconditions for hybridisation as described under the general methods. Thechip was then imaged using an orange filter with an exposure time of 1s.

Results

As expected, the channels grafted with the non-cleavable primers (chs 2and 3) treated or not treated with sodium periodate did not give anyhybridization signal with the sequencing primer. As the colonies inthese channels are still double-stranded no hybridization of thesequencing primer can occur.

No signal was observed for the cleavable primer treated with milliQwater (no sodium periodate).

Hybridization of the sequencing primer was detected in the two diolchannels treated with sodium periodate.

Example 4 Acrylamide Coating of Silex Flowcells

The solid supports used were typically 8-channel glass flowcells such asthose provided by Silex Microsystems (Sweden). However, the experimentalconditions and procedures are readily applicable to other solidsupports.

A schematic representation of one such flowcell is shown in FIG. 7. Theflowcell is formed in three layers. The bottom layer 1 is formed ofborosilicate glass at a depth of 1000 μm. An etched silicon channellayer (100 μm depth) is placed on top to defined 8 separate reactionchannels. Top layer 3 (300 μm depth) includes two separate series of 8holes 4 and 4′ in register with the channels of the etched siliconchannel layer in order to provide fluid communication with the contentsof the channels when the flowcell is assembled in use.

Flowcells were washed as follows: neat Decon for 30 min, milliQ H₂O for30 min, NaOH 1N for 15 min, milliQ H₂O for 30 min, HCl 0.1N for 15 min,milliQ H₂O for 30 min.

Polymer Solution Preparation

For 10 ml of 2% polymerisation mix.

-   -   10 ml of 2% solution of acrylamide in milliQ H₂O    -   165 μl of a 100 mg/ml N-(5-bromoacetamidylpentyl)acrylamide        (BRAPA) solution in DMF (23.5 mg in 235 μl DMF)    -   11.5 μl of TEMED    -   100 μl of a 50 mg/ml solution of potassium persulfate in milliQ        H₂O (20 mg in 400 μl H₂O)

The 10 ml solution of acrylamide was first degassed with argon for 15min. The solutions of BRAPA, TEMED and potassium persulfate weresuccessively added to the acrylamide solution. The mixture was thenquickly vortexed and immediately pumped into the flowcell.Polymerization was then carried out for 1 h 30 at RT. Afterwards thechannels were washed with milliQ H₂O for 30 min and filled with 0.1 Mpotassium phosphate buffer for storage until required.

Example 5 Grafting Primers onto Surface of Acrylamide Coated SilexFlowcell

An acrylamide (SFA) coated flowcell was placed onto a modifiedMJ-Research thermocycler and attached to a peristaltic pump. Graftingmix consisting of 0.5 μM of a forward primer and 0.5 μM of a reverseprimer in 10 mM phosphate buffer (pH 7.0) was pumped into the channelsof the flowcell at a flow rate of 60 μl/min for 75 s at 20° C. Thethermocycler was then heated up to 51.6° C., and the flowcell incubatedat this temperature for 1 hour. During this time, the grafting mixunderwent 18 cycles of pumping: grafting mix was pumped in at 15 μl/minfor 20 s, then the solution was pumped back and forth (5 s forward at 15μl/min, then 5 s backward at 15 μl/min) for 180 s. After 18 cycles ofpumping, the flowcell was washed by pumping in 5×SSC/5 mM EDTA at 15μl/min for 300 s at 51.6° C. The thermocycler was then cooled to 20° C.

The primers were typically 5′-phosphorothioate oligonucleotidesincorporating any specific sequences or modifications required forcleavage. Their sequences and suppliers varied according to theexperiment they were to be used for, and in this case were complementaryto the 5′-ends of the template duplex. For the experiment described, theamplified clusters contained a diol linkage in one of the graftedprimers. Diol linkages can be introduced by including a suitable linkageinto one of the primers used for solid-phase amplification.

The grafted primers contained a sequence of T bases at the 5′-end to actas a spacer group to aid linearisation and hybridization. Synthesis ofthe diol phosphoramidite is detailed below. Oligonucleotides wereprepared using the diol phosphoramidite using standard couplingconditions on a commercial DNA synthesiser. The finalcleavage/deprotection step in ammonia cleaves the acetate groups fromthe protected diol moiety, so that the oligonucleotide in solutioncontains the diol modification. The sequences of the two primers graftedto the flowcell are:

5′-PS-TTTTTTTTTT-Diol-AATGATACGGCGACCACCGA-3′(equivalent to SEQ ID NO: 3 but including diol linkage) And(SEQ ID NO: 5) 5′-PS-TTTTTTTTTTCAAGCAGAAGACGGCATACGA-3′

Example 6 Preparation of Diol-Phosphoramidite for DNA Coupling

Step 1:

1,6-Hexanediol (Sigma Aldrich 99%) (14.6 g, 124 mmol),N,N-diisopropylethylamine (Hünig's base; Sigma Aldrich; redistilled)(21.6 mL, 124 mmol) was dissolved in anhydrous DCM/DMF (250/50 mL) underN₂. The solution was cooled to 0° C. and the first portion of4,4′-dimethoxytrityl chloride (DMTr-Cl; Sigma-Aldrich 95%) (10.5 g, 31mmol) added. The reaction mixture was then warmed to room temperature.After stirring for 1 h, the reaction mixture was cooled to 0° C. againand the second portion of DMTr-Cl (10.5 g, 31 mmol) was added and thenallowed to stir at room temperature for other 2 hours. TLC (EtOAc:petroleum ether 4:6) analysis indicated ca. 95% consumption of startingmaterial derivative (DMTr-OH). The reaction was concentrated underreduced pressure and Aq. NaHCO₃ (sat.) solution (500 mL) was poured intothe residue. The resulting mixture was extracted with petroleumether/EtOAc (2:1) (3×1000 mL). The combined organic layers were driedover MgSO₄, and concentrated under vacuum. The residue was co-evaporatedwith xylene (2×100 mL) to remove DMF. The reaction mixture, waspre-absorbed on silica gel and subjected to flash chromatography usingsolvents containing 1% Et₃N petroleum ether to petroleum ether/EtOAc(7:3) as eluent. The yield of pale yellow oil was 16.58 g, 64%, with afurther 7.8 g (17%) of the bis-tritylated by-product.

TLC: R_(f): 0.35 (diol-1); R_(f): 0.7 (bis-tritylated by-product)(petroleum ether/EtOAc 6:4).

¹H NMR (400 MHz, CDCl₃): δ 1.32-1.44 (m, 4H, 2×CH₂), 1.54-1.68 (m, 4H,2×CH₂), 3.06 (t, J=6.6 Hz, 2H, CH₂O), 3.62-3.68 (m, 2H, CH₂OH), 3.81 (s,6H, 2× MeO), 6.83-6.85 (m, 4H, Ph), 7.24-7.35 (m, 7H, Ph), 7.45-7.47 (m,2H, Ph).

Step 2:

To a solution of Diol-1 (16.6 g, 39.5 mmol) in anhydrous DCM (200 mL),tetrapropylammonium perruthenate (TPAP; Sigma Aldrich 97%) (277 mg, 0.79mmol) was added under N₂ atmosphere. The solution was cooled to 0° C.and N-methylmopholine N-oxide (Sigma Aldrich 97%) (2.7 g, 23 mmol) wasadded. The reaction was warmed to room temperature. After 1 hour, theother three portions of N-methylmopholine N-oxide (3×2.0 g, 51.2 mmol)were added within a period of four hours. TLC (EtOAc: petroleum ether4:6) indicated that the reaction goes to completion. The reaction wasquenched with aq. NaHCO₃ (sat.) (1000 mL) and extracted to CH₂Cl₂(4×1000 mL). The combined organic layers were dried over MgSO₄. Thesolution was concentrated under reduced pressure. Diol-3, 9.9 g, 60%,was isolated by flash chromatography using solvents containing 1% Et₃Nfrom petroleum ether to petroleum ether/EtOAc (6:4) as eluent, as a paleyellow oil.

TLC: R_(f): 0.7 (petroleum ether/EtOAc 6:4).

¹H NMR (400 MHz, CDCl₃): δ 1.30-1.37 (m, 2H, CH₂), 1.48-1.57 (m, 4H,2×CH₂), 2.34 (td, J=1.7 and 7.4 Hz, 2H, CH₂CHO), 2.97 (s, 2H, CH₂O),3.72 (s, 6H, 2× MeO), 6.73-6.76 (m, 4H, Ph), 7.10-7.26 (m, 7H, Ph),7.34-7.36 (m, 2H, Ph), 9.67 (t, J=1.7, 1H, CHO).

Step 3:

A solution of triphenylphosphine (Sigma-Aldrich 99%, ReagentPlus™).(39.3 g, 150 mmol) and 4-bromobutyl acetate (Sigma-Aldrich) (26 mL, 180mmol) in anhydrous toluene (300 mL) was heated under reflux for 36 hoursunder N₂ in an oil-bath (140° C.). During the reflux, oil wasprecipitated out. The reaction mixture was cooled to room temperature.TLC (petroleum ether/EtOAc 7:3) analysis of the toluene solution showedthat there was still triphenylphosphine (R_(f): 0.8) left. Thesupernatant was decanted into another round-bottomed flask andconcentrated down to the approximate volume of 30 mL. The solution washeated under reflux again for another 12 hours. The supernatant wasdecanted. The portions of oil were combined together, dissolved in water(500 mL) and extracted with EtOAc (2×500 mL). The combined organiclayers were back-extracted with water (150 mL). Two lots of aqueouslayers were combined, evaporated under reduced pressure. The resultingresidue was co-evaporated with acetonitrile (2×100 mL) to give 78.4 g,95% yield of a pale yellow oil. NMR indicates that the product was pure,and was used for the next step reaction without further purification.

TLC: R_(f): 0.0 (petroleum ether/EtOAc 7:3).

¹H NMR (400 MHz, CDCl₃): δ 1.63-1.73 (m, 2H, CH₂), 1.94 (s, 3H, 2×CH₃),2.06-2.16 (m, 2H, CH₂), 3.97-4.05 (m, 2H, CH₂P), 4.11 (t, J=6.0, 2H,CH₂O), 7.69-7.95 (m, 15H, Ph).

³¹P NMR (162 MHz, CDCl₃): 25.9 ppm.

Mass spec details: LC-MS (Electrospray positive): (M⁺) 377.

Step 4:

Diol-2 (10.34 g, 22.7 mmol) was weighed into a round-bottomed flask anddissolved with DCM (20 mL). The solution was then evaporated underreduced pressure until it gave a white foam. The flask was thensubjected to high vacuum for 24 h. To this flask, anhydrous THF (180 mL)was added under N₂. The resulting suspension was cooled to −78° C. withan acetone-dry ice bath. With vigorous stirring, KOBu^(t) (3.3 g, 29.5mmol) was added under N₂. Slowly the colour of the suspension turnedorange, and white solids were gradually precipitated out. To thissuspension, a solution of diol-3 (dried at 60° C. under high vacuum for1 h before the reaction), (9.5 g, 22.7 mmol) in THF (50 mL) was addeddrop wise over half an hour. The acetone-dry ice bath was then removed.The reaction mixture was slowly warmed up to room temperature andstirred for another hour. The colour of the reaction mixture turned intoyellow after the addition of diol-3. The reaction mixture wasconcentrated down under reduced pressure. The resulting residue waspartitioned between DCM (800 mL) and aq. NaCl (sat.) (800 mL). Theaqueous layer was extracted with an additional DCM (2×800 mL). Theorganic extractions were combined, dried over MgSO₄, filtered, andevaporated under reduced pressure to give yellow oil. The oil wasdissolved in THF/MeOH (125/100 mL) and cooled to 0° C. To this solution,NaOH (1M in H₂O, 25 mL) was added. After allowing the reaction to stirfor 1 hour, TLC analysis indicated full consumption of startingmaterial. The reaction mixture was neutralized with acetic acid (1.5mL). The reaction mixture was concentrated down under reduced pressure.The resulting residue was partitioned between DCM (800 mL) and aq.NaHCO₃ (sat.) (800 mL). The aqueous layer was extracted with additionalDCM (2×800 mL). The organic extractions were combined, dried over MgSO₄,filtered, and evaporated to give a pale yellow oil. Diol-4, 6.45 g, 60%was isolated by flash chromatography using solvents containing 1% Et₃Nfrom petroleum ether to petroleum ether/EtOAc (5:5) as eluent, as alight yellow oil.

TLC: R_(f)=0.45 (petroleum ether/EtOAc 6:4).

¹H NMR (400 MHz, CDCl₃) δ 1.24-1.32 (m, 4H, 2×CH₂), 1.54-1.57 (m, 4H,2×CH₂), 1.93-1.96 (m, 2H, CH₂), 2.02-2.07 (m, 2H, CH₂), 2.96 (t, J=6.6Hz, 2H, CH₂O), 3.54-3.59 (m, 2H, CH₂OH), 3.72 (s, 6H, 2× MeO), 5.29-5.32(m, 2H, 2×=CH), 6.73-6.77 (m, 4H, Ph), 7.11-7.27 (m, 7H, Ph), 7.36-7.38(m, 2H, Ph).

Step 5:

To a solution of Diol-4 (5.68 g, 12 mmol) and imidazole (Sigma Aldrich,99%), (1.63 g, 24 mmol) in anhydrous DMF (100 mL), t-butyldiphenylsilylchloride (Sigma Aldrich, 98%), (4.05 mL, 15.6 mmol) was added drop wiseunder N₂ atmosphere at room temperature. The reaction was stirred for 1hour. TLC (petroleum ether/EtOAc 8:2) indicated that the startingmaterial was fully consumed. A saturated aq. NaHCO₃ solution (500 mL)was added to quench the reaction. The resulting mixture was extractedwith petroleum ether/EtOAc (2:1) (3×500 mL). The organic layers werecombined, dried over MgSO₄, filtered, and evaporated to give a yellowoil. Diol-5, 8.14 g, 95% was isolated by flash chromatography usingsolvents containing 1% Et₃N from petroleum ether to petroleumether/EtOAc (9:1) as eluent, as a colourless oil.

TLC: R_(f)=0.7 (petroleum ether:EtOAc 8:2).

¹H NMR (400 MHz, CDCl₃): δ 0.97 (s, 9H, 3×Me), 1.19-1.30 (m, 4H, 2×CH₂),1.48-1.55 (m, 4H, 2×CH₂), 1.91-1.95 (m, 2H, CH₂), 2.01-2.06 (m, 2H,CH₂), 2.95 (t, J=6.6 Hz, 2H, CH₂O), 3.58 (t, J=6.3 Hz, 2H, CH₂O), 3.70(s, 6H, 2× MeO), 5.24-5.27 (m, 2H, 2×=CH), 6.72-6.75 (m, 4H, Ph),7.11-7.37 (m, 15H, Ph), 7.57-7.60 (m, 4H, Ph).

Step 6:

A mixture of diol-5 (9.27 g, 13 mmol), AD-mix-α (Sigma Aldrich), (18.2g), methanesulfonamide (Sigma Aldrich, 97%), (1.23 g, 13 mmol), t-BuOH(65 mL) and water (65 mL) was stirred together vigorously at 55° C. for14 h. The TLC analysis (petroleum ether:EtOAc 6:4) indicated ca. 95%consumption of the starting material. The reaction mixture was cooled toroom temperature, treated with sodium sulfite (15.3 g, 12 mmol), thenfurther stirred for 30 min. A saturated aq. NaHCO₃ solution (500 mL) wasadded to the reaction. The resulting mixture was extracted with EtOAc(3×500 mL). The organic layers were combined, dried over MgSO₄,filtered, and evaporated to give yellow oil. Diol-6, 7.96 g, 82%, wasisolated by flash chromatography (silica gel, Fluka, 70-230 mesh) usingsolvents containing 1% Et₃N from petroleum ether to petroleumether/EtOAc (1:1) as elutant, as a white solid.

TLC: R_(f)=0.3 (petroleum ether:EtOAc 6:4).

¹H NMR (400 MHz, CDCl₃) δ 1.07 (s, 9H, 3×Me), 1.41-1.7 (m, 12H, 6×CH₂),1.94 (d, J=4.3 Hz, 1H, OH), 2.94-2.95 (m, 1H, OH), 3.06 (t, J=6.6 Hz,2H, CH₂O), 3.61-3.63 (m, 2H, 2× ChOH), 3.73 (t, J=5.6 Hz, 2H, CH₂O),3.81 (s, 6H, 2× MeO), 5.24-5.27 (m, 2H, 2×=CH), 6.82-6.85 (m, 4H, Ph),7.21-7.47 (m, 15H, Ph), 7.57-7.60 (m, 4H, Ph).

Step 7:

To a solution of diol-6 (7.96 g, 13 mmol) and DMAP (Sigma-AldrichReagentPlusm, 99%). (260 mg, 2.13 mmol) in a mixture of pyridine (15 mL)and DCM (30 mL), acetic anhydride (Fluka 99%), (2.5 mL, 26.68 mmol) wasadded at room temperature. TLC analysis (petroleum ether:EtOAc 6:4)indicated full consumption of the starting material after 1 h. Thereaction was quenched by saturated aq. NaHCO₃ solution (500 mL). After 5min. the mixture was extracted with DCM (3×500 mL). The organic layerswere combined, dried over MgSO₄, filtered, and evaporated. The residuewas co-evaporated with toluene (2×100 mL). The resulting yellow oil wassubjected to a plug of silica gel (50 g, Fluka, 70-230 mesh) to removeDMAP using eluents containing 0.1% Et₃N from petroleum ether topetroleum ether/EtOAc (7:3) (250 mL each). The combined fractions ofproduct were concentrated to dryness. The resulting colourless oil wasdissolved in THF (100 mL) and treated with TBAF (Sigma-Aldrich; 5% wtwater), (1 M in THF, 15 mL) at 0° C. The reaction solution was slowlywarmed up to room temperature and stirred for further 2 hours. TLCanalysis (petroleum ether:EtOAc 6:4) indicated that desilylation wascompleted. The volatile solvent (THF) was evaporated under reducedpressure at low temperature. A saturated aq. NaHCO₃ solution (500 mL)was added to the residue. The resulting mixture was extracted with EtOAc(3×500 mL). The organic layers were combined, dried over MgSO₄,filtered, and evaporated to give yellow oil. Diol-7, 4.2 g, 66%, wasisolated by flash chromatography using solvents containing 1% Et₃N frompetroleum ether to petroleum ether/EtOAc (1:1) as elutant, as a whitesolid.

TLC: R_(f)=0.45 (petroleum ether:EtOAc 1:1).

¹H NMR (400 MHz, CDCl₃) δ 1.29-1.33 (m, 4H, 2×CH₂), 1.47-1.63 (m, 8H,4×CH₂), 1.99, 2.01 (2s, 6H, 2 MeC(O)), 3.00 (t, J=6.5 Hz, 2H, CH₂O),3.60-3.64 (m, 2H, CH₂O), 3.75 (s, 6H, 2× MeO), 4.92-4.97 (m, 2H, 2×CHOAc), 6.76-6.80 (m, 4H, Ph), 7.15-7.29 (m, 7H, Ph), 7.38-7.40 (m, 2H,Ph).

Step 8:

To a solution of diol-7 (2.08 g, 3.5 mmol) and diisopropylethylamine(Sigma Aldrich), (1.53 ml, 8.75 mmol) in DCM (17 mL), 2-cyanoethylN,N-diisopropylchlorophosphor-amidite (1.0 g, 4.2 mmol) was added dropwise at room temperature under N₂. After stirring for 1 hour, TLCanalysis (petroleum ether:EtOAc 4:6) indicated full consumption of thestarting material. The solvent (THF) was concentrated under reducedpressure. The resulting residue was subjected to chromatographydirectly. Diol-8, 2.5 g, 90%, was isolated by flash chromatography usingsolvents containing 1% Et₃N from petroleum ether to petroleumether/EtOAc (1:1) as eluent, as a colourless syrup.

TLC: R_(f)=0.55 (petroleum ether:EtOAc 4:6).

¹H NMR (400 MHz, CDCl₃) δ 1.09, 1.10, 1.11, 1.12 (4 s, 12H, N(CHMe₂)₂),1.26-1.31 (m, 4H, 2×CH₂), 1.45-1.56 (m, 8H, 4×CH₂), 1.95, 1.969, 1.971,1.98 (4 s, 6H, 2 MeCO), 2.56 (t, J=6.5 Hz, 2H, CH₂CN), 2.95 (t, J=6.5Hz, 2H, CH₂O), 3.49-3.55 (m, 4H, CH₂O), 3.72 (s, 6H, 2× MeO), 4.89-4.92(m, 2H, 2× CHOAc), 6.74-6.76 (m, 4H, Ph), 7.13-7.25 (m, 7H, Ph),7.34-7.37 (m, 2H, Ph).

³¹P NMR (162 MHz, CDCl₃): 148.67, 148.69 ppm.

Example 7 Formation of DNA Colonies (Clusters) on Silex Chips Step 1:Hybridisation and Amplification

The DNA sequence used in the amplification process was a singlemonotemplate sequence of 364 bases, with ends complimentary to thegrafted primers. The full sequence of the template duplex is shown inFIG. 8 (SEQ ID NO:8). The duplex DNA (1 nM) was denatured using 0.1 Msodium hydroxide treatment followed by snap dilution to the desired0.2-2 μM ‘working concentration’ in ‘hybridization buffer’ (5×SSC/0.1%tween).

Surface amplification was carried out by thermocycling using an MJResearch thermocycler, coupled with an 8-way peristaltic pump IsmatecIPC ISM931 equipped with Ismatec tubing (orange/yellow, 0.51 mm ID).

The single stranded template was hybridised to the grafted primersimmediately prior to the amplification reaction, which thus begins withan initial primer extension step rather than template denaturation. Thehybridization procedure begins with a heating step in a stringent bufferto ensure complete denaturation prior to hybridisation.

After the hybridization, which occurs during a 20 min slow cooling step,the flowcell was washed for 5 minutes with a wash buffer (0.3×SSC/0.1%tween).

A typical amplification process is detailed in the following table,detailing the flow volumes per channel:

T Time Flow rate Pumped V Step Description (° C.) (sec) (μl/min) (μl) 1Pump Hybridization pre-mix 20 120 60 120 2 Pump Hybridization mix 98.5300 15 75 3 Remove bubbles 98.5 10 100  16.7 4 Stop flow and hold T 98.530 static 0 5 Slow cooling 98.5-40.2 19.5 min static 0 6 Pump washbuffer 40.2 300 15 75 7 Pump amplification pre-mix 40.2 200 15 50 8 Pumpamplification mix 40.2 75 60 75 9 First Extension 74 90 static 0 10 ampDenaturation 98.5 45 static 0 cycles Re-fill channels 98.5 10 60 10 1 to30 Annealing 58 90 static 0 Extension 74 90 static 0 11  Hold at 20° C.20 for ever static 0 12  Pump wash buffer 74 300 15 75Hybridisation pre mix (buffer)=5×SSC/0.1% tweenHybridisation mix=0.1 M hydroxide DNA sample, diluted in hybridisationpre mixWash buffer=0.3×SSC/0.1% tweenAmplification pre mix=2 M betaine, 20 mM Tris, 10 mM Ammonium Sulfate, 2mM Magnesium sulfate, 0.1% Triton, 1.3% DMSO, pH 8.8Amplification mix=2 M betaine, 20 mM Tris, 10 mM Ammonium Sulfate, 2 mMMagnesium sulfate, 0.1% Triton, 1.3% DMSO, pH 8.8 plus 200 μM dNTP mixand 25 units/mL of Taq polymerase (NEB Product ref M0273L)

Step 2: Linearisation

To linearize the nucleic acid clusters formed within the flow cellchannels, the computer component of the instrumentation flowed theappropriate linearization buffer through the flow cell for 20 mins atroom temp at 15 μL/min (total volume=300 μL per channel), followed bywater for 5 mins at r.t.

The linearisation buffer consisted of 1429 μL of water, 64 mg of sodiumperiodate, 1500 μL of formamide, 60 μL of 1 M Tris pH 8, and 11.4 μL of3-aminopropanol, mixed for a final volume of 3 mL. The periodate wasfirst mixed with the water while the Tris was mixed with the formamide.The two solutions were then mixed together and the 3-aminopropanol addedto that mixture.

Step 3: Blocking extendable 3′-OH groups

To prepare the blocking pre-mix, 1360 μL of water, 170 μL of 10×blocking buffer (NEB buffer 4; product number B7004S), and, 170 μL ofcobalt chloride (25 mM) were mixed for a final volume of 1700 μL. Toprepare the blocking mix 1065.13 μL of blocking pre-mix, 21.12 μL of 125μM ddNTP mix, and 13.75 μL of TdT terminal transferase (NEB; part noM0252S) were mixed for a final volume of 1100 μL.

To block the nucleic acid within the clusters formed in the flow cellchannels, the computer component of the instrumentation flowed theappropriate blocking buffer through the flow cell, and controlled thetemperature as shown in the exemplary embodiments below.

T Time Flow rate Pumped V Step Description (° C.) (sec) (μl/min) (μl) 1Pump Blocking 20 200 15 50 pre-mix 2 Pump Blocking mix 37.7 300 15 75 3Stop flow and 37.7  20 static 0 hold T 4 Cyclic pump 37.7 8 × 15/static45 Blocking mix and (20 + 180) wait 5 Pump wash buffer 20 300 15 75

Step 4: Denaturation and Hybridization of Sequencing Primer

To prepare the primer mix, 895.5 μL of hybridization pre-mix/buffer and4.5 μl of sequencing primer (100 μM) were mixed to a final volume of 900μL. The sequence of the sequencing primer used in this reaction was:

(SEQ ID NO: 7) 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC-3′

To denature the nucleic acid within the clusters and to hybridize thesequencing primer, the computer component of the instrumentation flowedthe appropriate solutions through the flow cell as described below:

T Time Flow rate Pumped V Step Description (° C.) (sec) (μl/min) (μl) 1Pump NaOH 20 300 15 75 2 Pump TE 20 300 15 75 3 Pump Primer mix 20 30015 75 4 Hold at 60 C 60 900 0 0 5 Pump wash buffer 40.2 300 15 75

After denaturation and hybridization of the sequencing primer, theflowcell was ready for sequencing.

Example 8 DNA Sequencing Cycles

Sequencing was carried out using modified nucleotides prepared asdescribed in International patent application WO 2004/018493, andlabelled with four different commercially available fluorophores(Molecular Probes Inc.).

A mutant 9° N polymerase enzyme (an exo-variant including the triplemutation L408Y/Y409A/P410V and C223S) was used for the nucleotideincorporation steps.

Incorporation mix, Incorporation buffer (50 mM Tris-HCl pH 8.0, 6 mMMgSO4, 1 mM EDTA, 0.05% (v/v) Tween −20, 50 mM NaCl) plus 110 nM YAVexo- C223S, and 1 μM each of the four labelled modified nucleotides, wasapplied to the clustered templates, and heated to 45° C.

Templates were maintained at 45° C. for 30 min, cooled to 20° C. andwashed with Incorporation buffer, then with 5×SSC/0.05% Tween 20.Templates were then exposed to Imaging buffer (100 mM Tris pH 7.0, 30 mMNaCl, 0.05% Tween 20, 50 mM sodium ascorbate, freshly dissolved).

Templates were scanned in 4 colours at room temp.

Templates were then exposed to sequencing cycles of Cleavage andIncorporation as follows:

Cleavage

-   -   Prime with Cleavage buffer (0.1 M Tris pH 7.4, 0.1 M NaCl and        0.05% Tween 20). Heat to 60° C.    -   Treat the clusters with Cleavage mix (100 mM TCEP in Cleavage        buffer).    -   Wait for a total of 15 min in addition to pumping fresh buffer        every 4 min.    -   Cool to 20° C.    -   Wash with Enzymology buffer.    -   Wash with 5×SSC/0.05% Tween 20.    -   Prime with Imaging buffer.    -   Scan in 4 colours at RT.

Incorporation

-   -   Prime with Incorporation buffer Heat to 60° C.    -   Treat with Incorporation mix. Wait for a total of 15 min in        addition to pumping fresh Incorporation mix every 4 min.    -   Cool to 20° C.    -   Wash with Incorporation buffer.    -   Wash with 5×SSC/0.05% Tween 20.    -   Prime with imaging buffer.    -   Scan in 4 colours at RT.    -   Repeat the process of Incorporation and Cleavage for as many        cycles as required.

Incorporated nucleotides were detected using a total internal reflectionbased fluorescent CCD imaging apparatus. Images were recorded andanalysed to measure the intensites and numbers of the fluorescentobjects on the surface. Images of two tiles from the first cycle ofnucleotide incorporation for a linearised and a non-linearised (noperiodate) channel are shown in FIG. 9. The histogram plots of thesetiles are shown in FIG. 10. In the case of the sequence selected foramplification, only a single base (T) was incorporated in each cluster.This figure clearly shows the presence of clusters in both thelinearised and non linearised channels, but the linearised clusters showa far higher average signal intensity. In the absence of thelinearisation step, the clusters remain double stranded, and can notundergo effective hybridisation with a sequencing primer, and thereforeshow greatly reduced signal upon treatment with nucleotides andpolymerase.

1.-38. (canceled)
 39. A method of generating a template for a nucleicacid sequencing reaction comprising, (i) providing a solid supportcomprising a plurality of amplification primers; (ii) amplifying atemplate using the primers on the support to produce a plurality ofdouble-stranded nucleic acid molecules, wherein both strands of themolecule are attached to the solid support at their 5′ ends; (iii)cleaving one strand of the double stranded molecules at a pre-determinedcleavage site, the cleavage not comprising cleavage with a restrictionendonuclease or a nicking endonuclease; (iv) subjecting the cleavedstrand to denaturing conditions to remove the portion of the cleavedstrand not attached to the solid support, thereby generating partiallyor substantially single-stranded templates for a nucleic acid sequencingreaction.
 40. The method of claim 39, wherein the double-strandednucleic acid molecules each comprise a target region flanked bynon-target sequences derived from the amplification primers used foramplification.
 41. The method of claim 40, wherein the pre-determinedcleavage site is not located within the target region of thedouble-stranded nucleic acid molecules.
 42. The method of claim 39,wherein cleavage comprises cleaving via a chemical cleavage reaction.43. The method of claim 39, wherein the pre-determined cleavage sitecomprises a diol linkage.
 44. The method of claim 39, wherein thepre-determined cleavage site comprises a uracil.
 45. The method of claim39, wherein the pre-determined cleavage site comprises one or moreribonucleotidies.
 46. The method of claim 39, wherein cleavage occurs byexposure to a metal ion.
 47. The method of claim 39, wherein thepre-determined cleavage site comprises one or more methylatednucleotides.
 48. The method of claim 39, wherein cleavage occurs by aphotochemical mechanism.
 49. The method of claim 39, whereinamplification of the template forms a cluster comprising the pluralityof double-stranded molecules.
 50. The method of claim 39, furthercomprising performing a sequencing reaction to determine the sequence ofat least one region of the template.
 51. The method of claim 50, whereinthe sequence of the target region is determined.
 52. The method of claim50, wherein the sequencing reaction comprises sequencing-by-synthesis.53. The method of claim 50, wherein the sequencing reaction comprisespyrosequencing or sequencing-by-ligation.
 54. The method of claim 39,wherein the solid support comprises a hydrogel.
 55. The method of claim54, wherein the hydrogel is a polyacrylamide hydrogel.
 56. The method ofclaim 54, wherein the solid support is prepared by a method comprisingpolymerizing on the solid support a mixture of: (i) a first comonomerwhich is acrylamide, methacrylamide, hydroxyethyl methacrylate orN-vinyl pyrrolidinone; and (ii) a second comonomer which is afunctionalized acrylamide or acrylate of formula (I):H₂C═C(H)—C(═O)-A-B-C  (I); or a methacrylate or methacrylamide offormula (II):H₂C═C(CH₃)—C(═O)-A-B-C  (II); wherein A is NR or O, wherein R ishydrogen or an optionally substituted saturated hydrocarbyl groupcomprising 1 to 5 carbon atoms; B is an optionally substituted alkylenebiradical of formula —(CHn)- wherein n is an integer from 1 to 50; andwherein n=2 or more, one or more optionally substituted ethylenebiradicals —CH2CH2- of said alkylene biradical may be independentlyreplaced by ethenylene and ethynylene moieties; and wherein n=1 or more,one or more methylene biradicals —CH2- may be replaced independentlywith an optionally substituted mono- or polycyclic hydrocarbon biradicalcomprising from 4 to 50 carbon atoms, or a correspondingdeteromonocyclic or heteropolycyclic biradical wherein at least 1 CH2 orCH2 is substituted by an oxygen sulfur or nitrogen atom or an NH group;and C of A-B-C of formulas (I) or (II) is a group for reaction with acompound to bind said compound covalently to said hydrogel to form apolymerized product, characterized in that polymerization is conductedon and immobilized the polymerized product to a solid support that isnot covalently surface-modified.